Activation of bioluminescence by structural complementation

ABSTRACT

Provided herein are compositions and methods for the assembly of a bioluminescent complex from two or more non-luminescent (e.g., substantially non-luminescent) peptide and/or polypeptide units. In particular, bioluminescent activity is conferred upon a non-luminescent polypeptide via structural complementation with another, complementary non-luminescent peptide.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. Provisional Patent Application Ser. No. 61/791,549 filed Mar. 15, 2013, which is hereby incorporated by reference in its entirety.

FIELD

Provided herein are compositions and methods for the assembly of a bioluminescent complex from two or more non-luminescent (e.g., substantially non-luminescent) peptide and/or polypeptide units. In particular, bioluminescent activity is conferred upon a non-luminescent polypeptide via structural complementation with another, complementary non-luminescent peptide.

BACKGROUND

Biological processes rely on covalent and non-covalent interactions between molecules, macromolecules and molecular complexes. In order to understand such processes, and to develop techniques and compounds to manipulate them for research, clinical and other practical applications, it is necessary to have tools available to detect and monitor these interactions. The study of these interactions, particularly under physiological conditions (e.g. at normal expression levels for monitoring protein interactions), requires high sensitivity.

SUMMARY

The present invention relates to compositions comprising complementary non-luminescent amino acid chains (e.g., substantially non-luminescent peptides and/or polypeptides that are not fragments of a preexisting protein), complexes thereof, and methods of generating an optically detectable bioluminescent signal upon association of the non-luminescent amino acid chains (e.g., peptides and/or polypeptides). In some embodiments, the present invention provides two or more non-luminescent, or substantially non-luminescent peptides and/or polypeptides, that, when brought together, assemble into a bioluminescent complex. In some embodiments, a pair of substantially non-luminescent peptide and/or polypeptide units assembles into a bioluminescent complex. In other embodiments, three or more substantially non-luminescent peptide and/or polypeptide units assemble into a bioluminescent complex (e.g., ternary complex, tertiary complex, etc.). Provided herein are technologies for detecting interactions between molecular entities (e.g., proteins, nucleic acids, carbohydrates, small molecules (e.g., small molecule libraries)) by correlating such interactions to the formation of a bioluminescent complex of otherwise non-luminescent (e.g., substantially non-luminescent) amino acid chains.

In some embodiments, the assembled pair catalyzes a chemical reaction of an appropriate substrate into a high energy state, and light is emitted upon return of the substrate to a more stable form. In some embodiments, a bioluminescent complex exhibits luminescence in the presence of substrate (e.g., coelenterazine, furimazine, etc.).

Although the embodiments described herein primarily describe and refer to complementary, non-luminescent amino acid chains that form bioluminescent complexes, it is noted that the present technology can equally be applied to other enzymatic activities. The embodiments described herein relating to luminescence should be viewed as applying to complementary, substantially non-enzymatically active amino acid chains (e.g., peptides and/or polypeptides that are not fragments of a preexisting protein), complexes thereof, and methods of generating an enzymatic activity upon association of the complementary, substantially non-enzymatically active amino acid chains (e.g., peptides and/or polypeptides). Further, embodiments described herein that refer to non-luminescent peptides and/or polypeptides are applied, in some embodiments, to substantially non-luminescent peptides and/or polypeptides.

The invention is further directed to assays for the detection of molecular interactions between molecules of interest by linking the interaction of a pair of non-luminescent peptides/polypeptides to the interaction molecules of interest (e.g., transient association, stable association, complex formation, etc.). In such embodiments, a pair of a non-luminescent elements are tethered (e.g., fused) to molecules of interest and assembly of the bioluminescent complex is operated by the molecular interaction of the molecules of interest. If the molecules of interest engage in a sufficiently stable interaction, the bioluminescent complex forms, and a bioluminescent signal is generated. If the molecules of interest fail to engage in a sufficiently stable interaction, the bioluminescent complex will not form or only form weakly, and a bioluminescent signal is not detectable or is substantially reduced (e.g., substantially undetectable, essentially not detectable, etc.). In some embodiments, the magnitude of the detectable bioluminescent signal is proportional (e.g., directly proportional) to the strength, favorability, and/or stability of the molecular interactions between the molecules of interest.

In some embodiments, the present invention provides peptides comprising an amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced when the peptide contacts a polypeptide consisting of SEQ ID NO: 440. In some embodiments, a detectable bioluminescent signal is produced when the peptide contacts a polypeptide having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 440. In certain embodiments, the detectable bioluminescent signal is produced, or is substantially increased, when the peptide associates with the polypeptide comprising or consisting of SEQ ID NO: 440, or a portion thereof. In preferred embodiments, the peptide exhibits alteration (e.g., enhancement) of one or more traits compared to a peptide of SEQ ID NO: 2, wherein the traits are selected from: affinity for the polypeptide consisting of SEQ ID NO: 440, expression, intracellular solubility, intracellular stability and bioluminescent activity when combined with the polypeptide consisting of SEQ ID NO: 440. Although not limited to these sequences, the peptide amino acid sequence may be selected from amino acid sequences of SEQ ID NOS: 3-438. In some embodiments, fusion polypeptides are provided that comprise: (a) an above described peptide, and (b) a first interaction polypeptide that forms a duplex with a second interaction polypeptide upon contact of the first interaction polypeptide and the second interaction polypeptide. In certain embodiments, bioluminescent complexes are provided that comprise: (a) a first fusion polypeptide described above and (b) a second fusion polypeptide comprising: (i) the second interaction polypeptide and (ii) a complement polypeptide that emits a detectable bioluminescent signal when associated with the peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2; wherein the first fusion polypeptide and second fusion polypeptide are associated; and wherein the peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2 and the complement polypeptide are associated.

In some embodiments, the present invention provides polypeptides comprising an amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 440, wherein a detectable bioluminescent signal is produced when the polypeptide contacts a peptide consisting of SEQ ID NO: 2. In some embodiments, a detectable bioluminescent signal is produced when the polypeptide contacts a peptide having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 2. In some embodiments, the polypeptide exhibits alteration (e.g., enhancement) of one or more traits compared to a peptide of SEQ ID NO: 440, wherein the traits are selected from: affinity for the peptide consisting of SEQ ID NO: 2, expression, intracellular solubility, intracellular stability, and bioluminescent activity when combined with the peptide consisting of SEQ ID NO: 2. Although not limited to such sequences, the polypeptide amino acid sequence may be selected from one of the amino acid sequences of SEQ ID NOS: 441-2156. In some embodiments, the detectable bioluminescent signal is produced when the polypeptide associates with the peptide consisting of SEQ ID NO: 2. In some embodiments, a fusion polypeptide is provided that comprises: (a) a polypeptide described above and (b) a first interaction polypeptide that forms a duplex with a second interaction polypeptide upon contact of the first interaction polypeptide and the second interaction polypeptide. In certain embodiments, a bioluminescent complex is provided that comprises: (a) a first fusion polypeptide described above; and (b) a second fusion polypeptide comprising: (i) the second interaction polypeptide and (ii) a complement peptide that causes the polypeptide comprising an amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 440 to emit a detectable bioluminescent signal when an association is formed between the two; wherein the first fusion polypeptide and second fusion polypeptide are associated; and wherein the polypeptide comprising an amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 440 and the complement peptide are associated.

In some embodiments, the present invention provides nucleic acids (e.g., DNA, RNA, etc.), oligonucleotides, vectors, etc., that code for any of the peptides, polypeptides, fusion proteins, etc., described herein. In some embodiments, a nucleic acid comprising or consisting of one of the nucleic acid sequences of SEQ ID NOS: 3-438 (coding for non-luminescent peptides) and/or SEQ ID NOS 441-2156 (coding for non-luminescent polypeptides) are provided. In some embodiments, other nucleic acid sequences coding for amino acid sequences of SEQ ID NOS: 3-438 and/or SEQ ID NOS 441-2156 are provided.

In certain embodiments, the present invention provides bioluminescent complexes comprising: (a) a peptide comprising a peptide amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 2; and (b) a polypeptide comprising a polypeptide amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 440, wherein the bioluminescent complex exhibits detectable luminescence. Although not limited to particular sequences, in some embodiments, the peptide amino acid sequence is selected from one of the amino acid sequences provided in SEQ ID NOS: 3-438.

In various embodiments, bioluminescent complexes are provided that comprise: (a) a first amino acid sequence that is not a fragment of a preexisting protein; and (b) a second amino acid sequence that is not a fragment of a preexisting protein, wherein the bioluminescent complex exhibits detectable luminescence, wherein the first amino acid sequence and the second amino acid sequence are associated, and wherein the bioluminescent complex emits a detectable bioluminescent signal when the first amino acid sequence and the second amino acid sequence are associated. Some such bioluminescent complexes further comprise: (c) a third amino acid sequence comprising a first member of an interaction pair, wherein the third amino acid sequence is covalently attached to the first amino acid sequence; and (d) a fourth amino acid sequence comprising a second member of an interaction pair, wherein the fourth amino acid sequence is covalently attached to the second amino acid sequence. In certain embodiments, interactions (e.g., non-covalent interactions (e.g., hydrogen bonds, ionic bonds, van der Waals forces, hydrophobic interactions, etc.) covalent interactions (e.g., disulfide bonds), etc.) between the first amino acid sequence and the second amino acid sequence do not significantly associate the first amino acid sequence and the second amino acid sequence in the absence of the interactions between the first member and the second member of the interaction pair. In some embodiments, a first polypeptide chain comprises the first amino acid sequence and the third amino acid sequence, and wherein a second polypeptide chain comprises the second amino acid sequence and the fourth amino acid sequence. In some embodiments, the first polypeptide chain and the second polypeptide chain are expressed within a cell.

In some embodiments, the present invention provides a bioluminescent complex comprising: (a) a pair of non-luminescent elements, wherein each non-luminescent element is not a fragment of a preexisting protein; (b) an interaction pair, wherein each interaction element of the interaction pair is covalently attached to one of the non-luminescent elements.

Various embodiments described herein provide methods of detecting an interaction between a first amino acid sequence and a second amino acid sequence comprising, for example, the steps of: (a) attaching the first amino acid sequence to a third amino acid sequence and attaching the second amino acid sequence to a fourth amino acid sequence, wherein the third and fourth amino acid sequences are not fragments of a preexisting protein, wherein a complex of the third and fourth amino acid sequences emits a detectable bioluminescent signal (e.g., substantially increased bioluminescence relative to the polypeptide chains separately), wherein the non-covalent interactions between the third and fourth amino acid sequences are insufficient to form, or only weakly form, a complex of the third and fourth amino acid sequences in the absence of additional stabilizing and/or aggregating forces, and wherein a interaction between the first amino acid sequence and the second amino acid sequence provides the additional stabilizing and/or aggregating forces to produce a complex of the third and fourth amino acid sequences; (b) placing the first, second, third, and fourth amino acid sequences of step (a) in conditions to allow for interactions between the first amino acid sequence and the second amino acid sequence to occur; and (c) detecting the bioluminescent signal emitted by the complex of the third and fourth amino acid sequences, wherein detection of the bioluminescent signal indicates an interaction between the first amino acid sequence and the second amino acid sequence. In some embodiments, attaching the first amino acid sequence to the third amino acid sequence and the second amino acid sequence to the fourth amino acid sequence comprises forming a first fusion protein comprising the first amino acid sequence and the third amino acid sequence and forming a second fusion protein comprising the second amino acid sequence and the fourth amino acid sequence. In some embodiments, the first fusion protein and the second fusion protein further comprise linkers between said first and third amino acid sequences and said second and fourth amino acid sequences, respectively. In certain embodiments, the first fusion protein is expressed from a first nucleic acid sequence coding for the first and third amino acid sequences, and the second fusion protein is expressed from a second nucleic acid sequence coding for the second and fourth amino acid sequences. In some embodiments, a single vector comprises the first nucleic acid sequence and the second nucleic acid sequence. In other embodiments, the first nucleic acid sequence and the second nucleic acid sequence are on separate vectors. In certain embodiments, the steps of (a) “attaching” and (b) “placing” comprise expressing the first and second fusion proteins within a cell.

Provided herein are methods of creating, producing, generating, and/or optimizing a pair of non-luminescent elements comprising: (a) aligning the sequences of three or more related proteins; (b) determining a consensus sequence for the related proteins; (c) providing first and second fragments of a protein related to three or more proteins (or providing first and second fragments of one of the three or more proteins), wherein the fragments are individually substantially non-luminescent but exhibit luminescence upon interaction of the fragments; (d) mutating the first and second fragments at one or more positions each, wherein the mutations alter the sequences of the fragments to be more similar to a corresponding portion of the consensus sequence (e.g., wherein the mutating results in a pair of non-luminescent elements that are not fragments of a preexisting protein), and (e) testing the pair of non-luminescent elements for the absence (e.g., essential absence, substantial absence, etc.) of luminescence when unassociated, and luminescence upon association of the non-luminescent pair into a bioluminescent complex. Examples of such a process are described in Examples 1-5. In some embodiments, the non-luminescent elements exhibit enhancement of one or more traits compared to the first and second fragments, wherein the traits are selected from: increased reconstitution affinity, decreased reconstitution affinity, enhanced expression, increased intracellular solubility, increased intracellular stability, and increased intensity of reconstituted luminescence.

In some embodiments, the present invention provides detection reagents comprising: (a) a polypeptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 440, wherein a detectable bioluminescent signal is produced when the polypeptide contacts a peptide consisting of SEQ ID NO: 2, and (b) a substrate for a bioluminescent complex produced by the polypeptide and a peptide consisting of SEQ ID NO: 2. In some embodiments, the present invention provides detection reagents comprising: (a) a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced when the peptide contacts a polypeptide consisting of SEQ ID NO: 440, and (b) a substrate for a bioluminescent complex produced by the peptide and a polypeptide consisting of SEQ ID NO: 440.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph depicting the effect of various mutations of the GVTGWRLCKRISA (SEQ ID NO: 236) peptide on luminescence resulting from complementation with SEQ ID NO: 440.

FIG. 2 shows a graph depicting the effect of various mutations of the SEQ ID NO: 440 polypeptide on luminescence resulting from complementation with GVTGWRLCKRISA (SEQ ID NO: 236) or GVTGWRLFKRISA (SEQ ID NO: 108) peptides.

FIG. 3A shows the luminescence (RLUs) detected in each non-luminescent polypeptide (NLpoly) mutant containing a single glycine to alanine substitution. FIG. 3B shows the fold increase in luminescence over wild-type.

FIG. 4A show the luminescence (RLUs) detected in each NLpoly mutant containing a composite of glycine to alanine substitutions. FIG. 4B shows the fold increase in luminescence over wild-type.

FIG. 5 shows a graph depicting the luminescence (RLUs) detected in HT-NLpeptide fusions.

FIG. 6 shows a graph depicting the luminescence (RLUs) detected in NLpeptide-HT fusions.

FIG. 7 shows a graph depicting the luminescence (RLUs) detected in NLpeptide-HT fusions.

FIG. 8 shows the luminescence (RLUs) generated by a luminescent complex after freeze-thaw cycles of non-luminescent peptide (NLpep).

FIG. 9 shows concentration normalized activity of peptides, and the TMR gel used to determine the relative concentrations.NLpep concentration FIG. 10 shows a graph of the luminescence of various mutations of residue R11 of NLpoly-5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).

FIG. 11 shows a graph of the luminescence of various mutations of residue A15 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).

FIG. 12 shows a graph of the luminescence of various mutations of residue L18 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).

FIG. 13 shows a graph of the luminescence of various mutations of residue F31 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).

FIG. 14 shows a graph of the luminescence of various mutations of residue V58 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).

FIG. 15 shows a graph of the luminescence of various mutations of residue A67 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).

FIG. 16 shows a graph of the luminescence of various mutations of residue M106 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).

FIG. 17 shows a graph of the luminescence of various mutations of residue L149 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).

FIG. 18 shows a graph of the luminescence of various mutations of residue V157 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).

FIG. 19 shows a graph of the luminescence of NLpep-HT fusions.

FIG. 20 shows a graph of the luminescence of NLpep-HT fusions, and a TMR gel indicating their relative expression levels.

FIG. 21 shows a graph of the luminescence of NLpep-HT fusions.

FIG. 22 shows a graph of the luminescence of NLpoly 5A2 (top) and NLpoly5A2+R11E in the presence of various NLpeps (bottom).

FIG. 23 shows a graph of the luminescence of NLpep-HT fusions.

FIG. 24 shows a graph of the luminescence of NLpolys 1-13 with NLpep53 (top) and without complimentary peptide (bottom).

FIG. 25 shows a graph of the luminescence of various NLpolys with NLpep53 with NANOGLO or DMEM buffer and furimazine or coelenterazine substrate.

FIG. 26 shows a graph comparing luminescence in the presence of a ratio of furimazine with coelenterazine for various NLpolys and NLpep53.

FIG. 27 shows a graph comparing luminescence in the presence of a ratio of furimazine to coelenterazine for various NLpolys and NLpep53.

FIG. 28 shows a graph comparing luminescence in the presence of furimazine with coelenterazine for various NLpolys and NLpep53 in HEK293 cell lysate.

FIG. 29 shows a graph of the luminescence of various combinations of NLpoly and NLpep pairs in DMEM buffer with furimazine.

FIG. 30 shows a graph of the signal/background luminescence of various combinations of NLpoly and NLpep pairs in DMEM buffer with furimazine.

FIG. 31 shows a graph of luminescence and substrate specificity of various NLpoly mutants with NLpep69 using either furimazine or coelenterazine as a substrate.

FIG. 32 shows a comparison of luminescence and substrate specificity of various NLpoly mutants with NLpep69 using either furimazine or coelenterazine as a substrate, and under either lytic (bottom graph) or live cell (top graph) conditions.

FIG. 33 shows a comparison of luminescence and substrate specificity of NLpoly mutants with NLpep78 using either furimazine or coelenterazine as a substrate, and under either lytic (bottom graph) or live cell (top graph) conditions.

FIG. 34 shows a comparison of luminescence and substrate specificity of various NLpoly mutants with NLpep79 using either furimazine or coelenterazine as a substrate, and under either lytic (bottom graph) or live cell (top graph) conditions.

FIG. 35 shows graphs of the luminescence of NLpep78-HT (top) and NLpep79-HT (bottom) fusions in the presence of various NLpolys.

FIG. 36 shows a graph of the luminescence of various NLpolys in the absence of NLpep.

FIG. 37 shows graphs of the luminescence of NLpep78-HT (top) and NLpep79-HT (bottom) fusions in the presence of various NLpolys with either furimazine or coelenterazine substrates.

FIG. 38 shows a graph of the luminescence of NLpep78-HT with various NLpolys expressed in CHO and HeLa cells.

FIG. 39 shows graphs of raw and normalized luminescence from NLpoly fused to firefly luciferase expressed in HEK293, Hela, and CHO cell lysates.

FIG. 40 shows graphs of raw and normalized luminescence from NLpoly fused to click beetle red luciferase expressed in HEK293, Hela, and CHO cell lysates.

FIG. 41 shows a graphs of luminescence of complementation in live cells using either NLpoly wild-type or 5P.

FIG. 42 shows graphs luminescence of cell-free complementation of NLpep78-HT fusion (top) and NLpep79-HT fusion (bottom) with various NLpolys.

FIG. 43 shows a graph of binding affinities for various combinations of NLpeps and NLpolys expressed in HeLa, HEK293 and CHO cell lysate.

FIG. 44 shows a graph of binding affinities for various combinations of NLpeps and NLpolys in PBS or NANOGLO buffer.

FIG. 45 shows a graph of binding affinities for NLpoly 5P with NLpep9 or NLpep53 expressed in HeLa, HEK293 or CHO cell lysate.

FIG. 46 shows a graph of luminescence of varying amounts of NLpolys in the absence of NLpep.

FIG. 47 shows a graph of background luminescence of various NLpoly variants.

FIG. 48 shows a graph of background luminescence of various NLpoly variants.

FIG. 49 shows a SDS-PAGE gel of total lysate and soluble fraction of several NLpoly variants

FIG. 50 shows (a) a SDS-PAGE gel of the total lysate and soluble fraction of NLpoly variants and (b) background luminescence of NLpoly variants.

FIG. 51 shows graphs of the luminescence generated with several NLpoly variants when complemented with 10 nm (right) or 100 nM (left) of NLpep78.

FIG. 52 shows graphs depicting background luminescence in E. coli lysate of various NLpoly variants.

FIG. 53 shows graphs depicting luminescence in E. coli lysate of various NLpoly variants complemented with NLpep78.

FIG. 54 shows graphs depicting luminescence in E. coli lysate of various NLpoly variants complemented with NLpep79.

FIG. 55 shows a graph of signal to background of various NLPolys complemented NLpoly variants complemented with NLpep78 or NLpep79 and normalized to NLpoly 5P.

FIG. 56 shows a graph depicting background, luminescence with NLpep79 (right) or NLpep78 (left) and signal-to-noise or various NLpoly variants.

FIG. 57 shows a SDS-PAGE gel of the total lysate and soluble fraction in various NLpoly 5P variants.

FIG. 58 shows (A) the amount of total lysate and soluble fraction of NLpoly 5P and NLpoly I107L, (B) luminescence generated by NLpoly 5P or NLpoly I107L without NLpep or with NLpep78 or NLpep79 and (C) the improved signal-to-background of NLpoly I107L over NLpoly 5P.

FIG. 59 shows graphs of luminescence for various NLpoly variants (A) without complementary peptide, (B) with NLpep78-HT and (C) with NLpep79-HT.

FIG. 60 shows graphs of luminescence for various NLpoly variants (A) without complementary peptide, (B) with NLpep78-HT and (C) with NLpep79-HT.

FIG. 61 shows graphs of luminescence for various NLpoly variants (A) without complementary peptide, (B) with NLpep78-HT and (C) with NLpep79-HT.

FIG. 62 shows graphs of luminescence for various NLpoly variants (A) without complementary peptide, (B) with NLpep78-HT and (C) with NLpep79-HT.

FIG. 63 shows binding affinity between an elongated NLpoly variant (additional amino acids at the C-terminus) and a shortened NLpep (deleted amino acids at the N-terminus).

FIG. 64 shows a graph of binding affinity of various NLpoly variants with NLpep78.

FIG. 65 shows the binding and Vmax of NLpep80 and NLpep87 to 5P expressed in mammalian cells (CHO, HEK293T and HeLa).

FIG. 66 shows the binding and Vmax of NLpep80 and NLpep87 to NLpoly 5P expressed in E. coli.

FIG. 67 shows a graph of luminescence of shortened NLpolys with elongated NLpeps.

FIG. 68 shows graphs of Kd and Vmax of NLpoly 5P in HeLa lysate with various complementary NLpeps.

FIG. 69 shows a graph of binding affinities for several NLpoly variants with NLpep81.

FIG. 70 shows a graph of binding affinities for several NLpoly variants with NLpep82.

FIG. 71 shows a graph of binding affinities for several NLpoly mutants with NLpep78.

FIG. 72 shows a graph of Michaelis constants for several NLpoly mutants with NLpep78.

FIG. 73 shows graphs of luminescence from a tertiary complementation of two NLpeps and NLpoly 5P-B9.

FIG. 74 shows a graph of luminescence of titration of NLpoly 5P with NLpep88-HT.

FIG. 75 shows images of intracellular localization of various NLpep fusions with HaloTag (HT).

FIG. 76 shows images of intracellular localization NLpoly(wt) and NLpoly(5P).

FIG. 77 demonstrates the ability to detect via complementation an NLPep-conjugated protein of interest following separation by SDS-PAGE and transfer to a PVDF membrane.

FIG. 78 shows a graph of relative luminescent signal from various NLpoly variants compared to NLpoly 5P (in the absence of NLpep).

FIG. 79 shows a graph of relative luminescent signal over background from various NLpolys compared to NLpoly 5P (in the absence of NLpep).

FIG. 80 compares the dissociation constants for NLpeps consisting of either 1 or 2 repeat units of NLpep78.

FIG. 81 shows the affinity between NLpoly 5A2 and NLpep86.

FIG. 82 shows graphs of the luminescence from NLpoly variants without NLpep, with NLpep78, and NLpep79.

FIG. 83-90 show the dissociation constants as well as the Vmax values for NLpoly 5A2, 5P, 8S and 11S with 96 variants of NLpeps.

FIG. 91 shows an image of a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants.

FIG. 92 shows an image of a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants as well as a table containing the dissociation constants for the same variants.

FIG. 93 shows the substrate specificity for NLpoly 5P and 11S with NLpep79 and demonstrates that NLpoly 11S has superior specificity for furimazine than 5P.

FIG. 94 shows an image of a protein gel that follows the affinity purification of NLpoly 8S through binding NLpep78.

FIG. 95 contains a table of the association and dissociation rate constants for the binding of NLpoly WT or 11S to NLpepWT, 78 or 79.

FIG. 96 shows the Km values for various pairs of NLpoly/NLpep.

FIG. 97 compares the dissociation constant for NLpoly 11S/NLpep79 at sub-saturating and saturating concentrations of furimazine.

FIG. 98 compares the Km values for NLpoly 5A2 with NLpepWT, 78 and 79.

FIG. 99 shows the luminescence of NLpolys from various steps in the evolution process in the absence of NLpep.

FIG. 100 shows the improvement in luminescence from E. coli-derived NLpoly over the course of the evolution process with an overall ˜10⁵ improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

FIG. 101 shows the improvement in luminescence from HeLa-expressed NLpoly over the course of the evolution process with an overall ˜10⁵ improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

FIG. 102 shows the improvement in luminescence from HEK293 cell-expressed NLpoly over the course of the evolution process with an overall ˜10⁴ improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

FIG. 103 shows dissociation constants and demonstrates a ˜10⁴ fold improvement in binding affinity from NLpolyWT:NLpepWT to NLpoly11S:NLpep86.

FIG. 104 shows an image of a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants from various steps of the evolution process.

FIG. 105 shows luminescence of various NLpolys in the absence of NLpep and in the presence of NLpep78 and NLpep79.

FIG. 106 shows luminescence of various NLpolys in the absence of NLpep and in the presence of NLpep78 and NLpep79.

FIG. 107 shows luminescence of various NLpolys in the absence of NLpep and in the presence of NLpep78 and NLpep79.

FIG. 108 shows a comparison of luminescence generated by cells expressing different combinations of FRB and FKBP fused to NLpoly5P and NLpep80/87 after 15 min treatment with rapamycin or vehicle. Fold induction refers to signal generated in the presence of rapamycin compared to signal generated with vehicle.

FIG. 109 shows a comparison of luminescence generated by cells expressing different combinations of FRB and FKBP fused to NLpoly5P and NLpep80/87 after 60 min treatment with rapamycin or vehicle.

FIG. 110 shows a comparison of luminescence generated by cells expressing different combinations of FRB and FKBP fused to NLpoly5P and NLpep80/87 after 120 min treatment with rapamycin or vehicle.

FIG. 111 shows a comparison of luminescence generated by cells expressing different combinations of FRB and FKBP fused to NLpoly5P and NLpep80/87 after 120 min treatment with rapamycin or vehicle. All 8 possible combinations of FRB and FKBP fused to NLpoly/NLpep were tested and less total DNA was used.

FIG. 112 shows a comparison of luminescence generated by FRB or FKBP fusions expressed in the absence of binding partner.

FIG. 113 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpoly5P and FKBP-NLpep80/87 DNA.

FIG. 114 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpoly5P or FKBP-NLpep80/87 DNA in the absence of binding partner.

FIG. 115 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpoly5P and FKBP-NLpep80/87 DNA. This example differs from FIG. 113 in that lower levels of DNA were used.

FIG. 116 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpoly5P or FKBP-NLpep80/87 DNA in the absence of binding partner. This differs from FIG. 114 in that lower levels of DNA were used.

FIG. 117 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpoly5P and FKBP-NLpep80 DNA after treatment with rapamycin for different lengths of time.

FIG. 118 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpoly5P and FKBP-NLpep87 DNA after treatment with rapamycin for different lengths of time.

FIG. 119 shows a comparison of luminescence generated by cells expressing different combinations of FRB-NLpoly5P with FKBP-NLpep80/87/95/96/97. Assay was performed in both a two-day and three-day format.

FIG. 120 shows a comparison of luminescence generated by cells expressing different combinations of FRB-NLpoly5A2 with FKBP-NLpep80/87/95/96/97. Assay was performed in both a two-day and three-day format.

FIG. 121 shows a comparison of luminescence generated by cells expressing different combinations of FRB-NLpoly5A2 or FRB-NLpoly11S with FKBP-NLpep101/104/105/106/107/108/109/110.

FIG. 122 shows a comparison of luminescence generated by cells transfected with different combinations of FRB-NLpoly5A2 or FRB-NLpoly11S with FKBP-NLpep87/96/98/99/100/101/102/103.

FIG. 123 shows a comparison of luminescence generated by cells transfected with different levels of FRB-NLpoly11S and FKBP-NLpep87/101/102/107 DNA.

FIG. 124 shows a comparison of luminescence generated by cells transfected with different levels of FRB-NLpoly5A2 and FKBP-NLpep87/101/102/107 DNA.

FIG. 125 shows a rapamycin dose response curve showing luminescence of cells expressing FRB-NLpoly5P and FKBP-NLpep80/87 DNA.

FIG. 126 shows a rapamycin dose response curve showing luminescence of cells expressing FRB-NLpoly5A2 or FRB-NLply11S and FKBP-NLpep87/101 DNA.

FIG. 127 shows a comparison of luminescence generated by cells expressing FRB-11S and FKBP-101 and treated with substrate PBI-4377 or furimazine.

FIG. 128 shows a rapamycin time course of cells expressing FRB-NLpoly11S/5A2 and FKBP-NLpep87/101 conducted in the presence or absence of rapamycin wherein the rapamycin was added manually.

FIG. 129 shows a rapamycin time course of cells expressing FRB-NLpoly11S/5A2 and FKBP-NLpep87/101 conducted in the presence or absence of rapamycin wherein the rapamycin was added via instrument injector.

FIG. 130 shows luminescence generated by FRB-NLpoly11S and FKBP-NLpep101 as measured on two different luminescence-reading instruments.

FIG. 131 provides images showing luminescence of cells expressing FRB-NLpoly11S and FKBP-NLpep101 at various times after treatment with rapamycin.

FIG. 132 provides a graph showing Image J quantitation of the signal generated by individual cells expressing FRB-NLpoly11S and FKBP-NLpep101 at various times after treatment with rapamycin.

FIG. 133 shows a comparison of luminescence in different cell lines expressing FRB-NLpoly11S and FKBP-NLpep101.

FIG. 134 shows a comparison of luminescence generated by cells expressing FRB-NLpoly11S and FKBP-NLpep101 after treatment with the rapamycin competitive inhibitor FK506.

FIG. 135 shows (left side) luminescence generated by cells expressing FRB-NLpoly11S and FKBP-NLpep101 after treatment with the rapamycin competitive inhibitor FK506, and (right side) the percent of luminescence remaining after treatment with FK506.

FIG. 136 shows luminescence generated by cells transfected with different combinations of V2R-NLpoly5A2 or V2R-NLpoly11S with NLpep87/101-ARRB2 in the presence or absence of the V2R agonist AVP.

FIG. 137 shows an AVP treatment time course showing luminescence generated by cells transfected with V2R-NLpoly11S and NLpep87/101-ARRB2 after treatment with AVP wherein AVP was added manually.

FIG. 138 shows an AVP treatment time course showing luminescence generated by cells transfected with different combinations of V2R-NLpoly5A2 or V2R-NLpoly11S with NLpep87/101-ARRB2 after treatment with AVP wherein AVP was added via instrument injector.

FIG. 139 shows an AVP treatment time course at 37° C. showing luminescence generated by cells expressing different configurations of V2R and ARRB2 fused to NLpoly11S and NLpep 101 after treatment with AVP.

FIG. 140 shows a comparison of luminescence in different cell lines expressing V2R-NLpep11S and NLpep101-ARRB2.

FIG. 141 shows 60× images showing luminescence of cells expressing V2R-NLpoly11S and NLpep101-ARRB2 at various times after treatment with AVP.

FIG. 142 shows 150× images showing luminescence of cells expressing V2R-NLpoly11S and NLpep101-ARRB2 at various times after treatment with AVP.

FIG. 143 shows a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants.

FIG. 144 shows the dissociation constants for NLpoly 5P and combinations of mutations at positions 31, 46, 75, 76, and 93 in NLpoly 5P.

FIG. 145 shows a transferase example of post translational modification enzyme activity detection using an NLpep and aminopeptidase.

FIG. 146 shows a hydrolase example of post translational modification enzyme activity detection using an NLpep and methyl-specific antibody.

FIG. 147 contains wavelength scans for NLpoly WT complemented with either NLpepWT or NLpepWT conjugated to TMR.

FIG. 148 contains wavelength scans for NanoLuc fused to HaloTag (NL-HT) and NLpoly 5A2 complemented with NLPepWT with 4 additional amino acids (DEVD) and conjugated to Non-chloroTOM (NCT).

FIG. 149 shows a schematic a tertiary interaction wherein the energy transfer with an NLpoly and NLpep can also be used to measure three molecules interacting. In the schematic, a GPCR labeled with an NLpoly and a GPCR interacting protein labeled with a NLpep form a bioluminescent complex when they interact. This allows measurement of the binary interaction. If a small molecule GPCR ligand bearing an appropriate fluorescent moiety for energy transfer interacts with this system, energy transfer will occur. Therefore, the binary protein-protein interaction and the ternary drug-protein-protein interaction can be measured in the same experiment.

DEFINITIONS

As used herein, the term “substantially” means that the recited characteristic, parameter, and/or value need not be achieved exactly, but that deviations or variations, including for example, tolerances, measurement error, measurement accuracy limitations and other factors known to skill in the art, may occur in amounts that do not preclude the effect the characteristic was intended to provide. A characteristic or feature that is substantially absent (e.g., substantially non-luminescent) may be one that is within the noise, beneath background, or below the detection capabilities of the assay being used.

As used herein, the term “bioluminescence” refers to production and emission of light by a chemical reaction catalyzed by, or enabled by, an enzyme, protein, protein complex, or other biomolecule (e.g., bioluminescent complex). In typical embodiments, a substrate for a bioluminescent entity (e.g., bioluminescent protein or bioluminescent complex) is converted into an unstable form by the bioluminescent entity; the substrate subsequently emits light as it returns to its more stable form.

As used herein the term “complementary” refers to the characteristic of two or more structural elements (e.g., peptide, polypeptide, nucleic acid, small molecule, etc.) of being able to hybridize, dimerize, or otherwise form a stable complex with each other. For example, a “complementary peptide and polypeptide” are capable of coming together to form a stable complex. Complementary elements may require assistance to form a stable complex (e.g., from interaction elements), for example, to place the elements in the proper conformation for complementarity, to co-localize complementary elements, to lower interaction energy for complementary, etc.

As used herein, the term “complex” refers to an assemblage or aggregate of molecules (e.g., peptides, polypeptides, etc.) in direct and/or indirect contact with one another. In one aspect, “contact,” or more particularly, “direct contact” means two or more molecules are close enough so that attractive noncovalent interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, and the like, dominate the interaction of the molecules. In such an aspect, a complex of molecules (e.g., a peptide and polypeptide) is stable under assay conditions such that the complex is thermodynamically more favorable than a non-aggregated, or non-complexed, state of its component molecules. As used herein the term “complex,” unless described as otherwise, refers to the assemblage of two or more molecules (e.g., peptides, polypeptides or a combination thereof).

As used herein, the term “non-luminescent” refers to an entity (e.g., peptide, polypeptide, complex, protein, etc.) that exhibits the characteristic of not emitting energy as light in the visible spectrum (e.g., in the presence or absence of a substrate). An entity may be referred to as non-luminescent if it does not exhibit detectable luminescence in a given assay. As used herein, the term “non-luminescent” is synonymous with the term “substantially non-luminescent.” An entity is “non-luminescent” if any light emission is sufficiently minimal so as not to create interfering background for a particular assay.

As used herein, the terms “non-luminescent peptide” (NLpep) and “non-luminescent polypeptide” (NLpoly) refer to peptides and polypeptides that exhibit substantially no luminescence (e.g., in the presence or absence of a substrate), or an amount that is virtually undetectable (e.g., beneath the noise) under standard conditions (e.g., physiological conditions, assay conditions, etc.) and with typical instrumentation (e.g., luminometer, etc.). In some embodiments, such non-luminescent peptides and polypeptides assemble, according to the criteria described herein, to form a bioluminescent complex. As used herein, a “non-luminescent element” is a non-luminescent peptide or non-luminescent polypeptide. The term “bioluminescent complex” refers to the assembled complex of two or more non-luminescent peptides and/or non-luminescent polypeptides. The bioluminescent complex catalyzes or enables the conversion of a substrate for the bioluminescent complex into an unstable form; the substrate subsequently emits light as it returns to its more stable form. When uncomplexed, two non-luminescent elements that form a bioluminescent complex may be referred to as a “non-luminescent pair.” If a bioluminescent complex is formed by three or more non-luminescent peptides and/or non-luminescent polypeptides, the uncomplexed constituents of the bioluminescent complex may be referred to as a “non-luminescent group.”

As used herein, the term “interaction element” refers to a moiety that assists in bringing together a pair of non-luminescent elements or a non-luminescent group to form a bioluminescent complex. In a typical embodiment, a pair of interaction elements (a.k.a. “interaction pair”) is attached to a pair of non-luminescent elements (e.g., non-luminescent peptide/polypeptide pair), and the attractive interaction between the two interaction elements facilitates formation of the bioluminescent complex; although the present invention is not limited to such a mechanism, and an understanding of the mechanism is not required to practice the invention. Interaction elements may facilitate formation of the bioluminescent complex by any suitable mechanism (e.g., bringing non-luminescent pair/group into close proximity, placing a non-luminescent pair/group in proper conformation for stable interaction, reducing activation energy for complex formation, combinations thereof, etc.). An interaction element may be a protein, polypeptide, peptide, small molecule, cofactor, nucleic acid, lipid, carbohydrate, antibody, etc. An interaction pair may be made of two of the same interaction elements (i.e. homopair) or two different interaction elements (i.e. heteropair). In the case of a heteropair, the interaction elements may be the same type of moiety (e.g., polypeptides) or may be two different types of moieties (e.g., polypeptide and small molecule). In some embodiments, in which complex formation by the interaction pair is studied, an interaction pair may be referred to as a “target pair” or a “pair of interest,” and the individual interaction elements are referred to as “target elements” (e.g., “target peptide,” “target polypeptide,” etc.) or “elements of interest” (e.g., “peptide of interest,” “polypeptide or interest,” etc.).

As used herein, the term “preexisting protein” refers to an amino acid sequence that was in physical existence prior to a certain event or date. A “peptide that is not a fragment of a preexisting protein” is a short amino acid chain that is not a fragment or sub-sequence of a protein (e.g., synthetic or naturally-occurring) that was in physical existence prior to the design and/or synthesis of the peptide.

As used herein, the term “fragment” refers to a peptide or polypeptide that results from dissection or “fragmentation” of a larger whole entity (e.g., protein, polypeptide, enzyme, etc.), or a peptide or polypeptide prepared to have the same sequence as such. Therefore, a fragment is a subsequence of the whole entity (e.g., protein, polypeptide, enzyme, etc.) from which it is made and/or designed. A peptide or polypeptide that is not a subsequence of a preexisting whole protein is not a fragment (e.g., not a fragment of a preexisting protein). A peptide or polypeptide that is “not a fragment of a preexisting bioluminescent protein” is an amino acid chain that is not a subsequence of a protein (e.g., natural of synthetic) that: (1) was in physical existence prior to design and/or synthesis of the peptide or polypeptide, and (2) exhibits substantial bioluminescent activity.

As used herein, the term “subsequence” refers to peptide or polypeptide that has 100% sequence identify with another, larger peptide or polypeptide. The subsequence is a perfect sequence match for a portion of the larger amino acid chain.

As used herein, the term “sequence identity” refers to the degree two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics and can be grouped into the families, e.g., acidic (e.g., aspartate, glutamate), basic (e.g., lysine, arginine, histidine), non-polar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.

As used herein, the term “physiological conditions” encompasses any conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, chemical makeup, etc. that are compatible with living cells.

As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Sample may also refer to cell lysates or purified forms of the peptides and/or polypeptides described herein. Cell lysates may include cells that have been lysed with a lysing agent or lysates such as rabbit reticulocyte or wheat germ lysates. Sample may also include cell-free expression systems. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention.

As used herein, unless otherwise specified, the terms “peptide” and “polypeptide” refer to polymer compounds of two or more amino acids joined through the main chain by peptide amide bonds (—C(O)NH—). The term “peptide” typically refers to short amino acid polymers (e.g., chains having fewer than 25 amino acids), whereas the term “polypeptide” typically refers to longer amino acid polymers (e.g., chains having more than 25 amino acids).

DETAILED DESCRIPTION

The study of protein interactions, particularly under physiological conditions and/or at physiologic expression levels, requires high sensitivity. In particular embodiments described herein, protein interactions with small molecules, nucleic acids, other proteins, etc. are detected based on the association of two non-luminescent elements that come together to from a bioluminescent complex capable of producing a detectable signal (e.g., luminescence). The formation of the bioluminescent complex is dependent upon the protein interaction that is being monitored.

Provided herein are compositions and methods for the assembly of a bioluminescent complex from two or more non-luminescent peptide and/or polypeptide units (e.g., non-luminescent pair). In some embodiments, the non-luminescent peptide and/or polypeptide units are not fragments of a preexisting protein (e.g., are not complementary subsequences of a known polypeptide sequence). In particular, bioluminescent activity is conferred upon a non-luminescent polypeptide via structural complementation with a non-luminescent peptide.

In some embodiments, provided herein are non-luminescent pairs for use in detecting and monitoring molecular interactions (e.g., protein-protein, protein-DNA, protein-RNA interactions, RNA-DNA, protein-small molecule, RNA-small-molecule, etc.). Also provided herein are complementary panels of interchangeable non-luminescent elements (e.g., peptides and polypeptides) that have variable affinities and luminescence upon formation of the various bioluminescent complexes (e.g., a high-affinity/high-luminescence pair, a moderate-affinity/high-luminescence pair, a low-affinity/moderate-luminescence pair, etc.). Utilizing different combinations of non-luminescent elements provides an adaptable system comprising various pairs ranging from lower to higher affinities, luminescence and other variable characteristics. This adaptability allows the detection/monitoring of molecular interactions to be fine-tuned to the specific molecule(s) of interest and expands the range of molecular interactions that can be monitored to include interactions with very high or low affinities. Further provided herein are methods by which non-luminescent pairs (or groups) and panels of non-luminescent pairs (or groups) are developed and tested.

In some embodiments, the interaction between the peptide/polypeptide members of the non-luminescent pair alone is insufficient to form the bioluminescent complex and produce the resulting bioluminescent signal. However, if an interaction element is attached to each peptide/polypeptide member of the non-luminescent pair, then the interactions of the interaction pair (e.g., to form an interaction complex) facilitate formation of the bioluminescent complex. In such embodiments, the bioluminescent signal from the bioluminescent complex (or the capacity to produce such a signal in the presence of substrate) serves as a reporter for the formation of the interaction complex. If an interaction complex is formed, then a bioluminescent complex is formed, and a bioluminescent signal is detected/measured/monitored (e.g., in the presence of substrate). If an interaction complex fails to form (e.g., due to unfavorable conditions, due to unstable interaction between the interaction elements, due to incompatible interaction elements), then a stable bioluminescent complex does not form, and a bioluminescent signal is not produced.

In certain embodiments, the interaction pair comprises two molecules of interest (e.g., proteins of interest). For example, assays can be performed to detect the interaction of two molecules of interest by tethering each one to a separate member of a non-luminescent pair. If the molecules of interest interact (e.g., transiently interact, stably interact, etc.), the non-luminescent pair is brought into close proximity in a suitable conformation and a bioluminescent complex is formed (and bioluminescent signal is produced/detected (in the presence of substrate)). In the absence of an interaction between the molecules of interest (e.g., no complex formation, not even transient interaction, etc.), the non-luminescent pair does not interact in a stable enough manner, and a bioluminescent signal is not produced or only weakly produced. Such embodiments can be used to study the effect of inhibitors on complex formation, the effect of mutations on complex formation, the effect of conditions (e.g., temperature, pH, etc.) on complex formation, the interaction of a small molecule (e.g., potential therapeutic) with a target molecule, etc.

Different non-luminescent pairs may require different strength, duration and/or stability of the interaction complex to result in bioluminescent complex formation. In some embodiments, a stable interaction complex is required to produce a detectable bioluminescent signal. In other embodiments, even a weak or transient interaction complex results in bioluminescent complex formation. In some embodiments, the strength of an interaction complex is directly proportional to the strength of the resulting bioluminescent signal. Some non-luminescent pairs produce a detectable signal when combined with an interaction complex with a high millimolar dissociation constant (e.g., K_(d)>100 mM). Other non-luminescent pairs require an interaction pair with a low millimolar (e.g., K_(d)<100 mM), micromolar (e.g., K_(d)<1 mM), nanomolar (e.g., K_(d)<1 μM), or even picomolar (e.g., K_(d)<1 nM) dissociation constant in order to produce a bioluminescent complex with a detectable signal.

In some embodiments, one or more of the non-luminescent peptides/polypeptides are not fragments of a pre-existing protein. In some embodiments, one or more of the non-luminescent peptides/polypeptides are not fragments of a pre-existing bioluminescent protein. In some embodiments, neither/none of the non-luminescent peptides/polypeptides are fragments of a pre-existing protein. In some embodiments, neither/none of the non-luminescent peptides/polypeptides are fragments of a pre-existing bioluminescent protein. In some embodiments, neither the non-luminescent peptide nor non-luminescent polypeptide that assemble together to form a bioluminescent complex are fragments of a pre-existing protein. In some embodiments, a non-luminescent element for use in embodiments of the present invention is not a subsequence of a preexisting protein. In some embodiments, a non-luminescent pair for use in embodiments described herein does not comprise complementary subsequences of a preexisting protein.

In some embodiments, non-luminescent peptides/polypeptides are substantially non-luminescent in isolation. In certain embodiments, when placed in suitable conditions (e.g., physiological conditions), non-luminescent peptides/polypeptides interact to form a bioluminescent complex and produce a bioluminescent signal in the presence of substrate. In other embodiments, without the addition of one or more interaction elements (e.g., complementary interaction elements attached to the component non-luminescent peptide and non-luminescent polypeptide), non-luminescent peptides/polypeptides are unable to form a bioluminescent complex or only weakly form a complex. In such embodiments, non-luminescent peptides/polypeptides are substantially non-luminescent in each other's presence alone, but produce significant detectable luminescence when aggregated, associated, oriented, or otherwise brought together by interaction elements. In some embodiments, without the addition of one or more interaction elements (e.g., complementary interaction elements attached to the component peptide and polypeptide), peptides and/or polypeptides that assemble into the bioluminescent complex produce a low level of luminescence in each other's presence, but undergo a significant increase in detectable luminescence when aggregated, associated, oriented, or otherwise brought together by interaction elements.

In some embodiments, compositions and methods described herein comprise one or more interaction elements. In a typical embodiment, an interaction element is a moiety (e.g., peptide, polypeptide, protein, small molecule, nucleic acid, lipid, carbohydrate, etc.) that is attached to a peptide and/or polypeptide to assemble into the bioluminescent complex. The interaction element facilitates the formation of a bioluminescent complex by any suitable mechanism, including: interacting with one or both non-luminescent elements, inducing a conformational change in a non-luminescent element, interacting with another interaction element (e.g., an interaction element attached to the other non-luminescent element), bringing non-luminescent elements into close proximity, orienting non-luminescent elements for proper interaction, etc.

In some embodiments, one or more interaction elements are added to a solution containing the non-luminescent elements, but are not attached to the non-luminescent elements. In such embodiments, the interaction element(s) interact with the non-luminescent elements to induce formation of the bioluminescent complex or create conditions suitable for formation of the bioluminescent complex. In other embodiments, a single interaction element is attached to one member of a non-luminescent pair. In such embodiments, the lone interaction element interacts with one or both of the non-luminescent elements to create favorable interactions for formation of the bioluminescent complex. In typical embodiments of the present invention, one interaction element is attached to each member of a non-luminescent pair. Favorable interactions between the interaction elements facilitate interactions between the non-luminescent elements. The interaction pair may stably interact, transiently interact, form a complex, etc. The interaction of the interaction pair facilitates interaction of the non-luminescent elements (and formation of a bioluminescent complex) by any suitable mechanism, including, but not limited to: bringing the non-luminescent pair members into close proximity, properly orienting the non-luminescent pair members from interaction, reducing non-covalent forces acting against non-luminescent pair interaction, etc.

In some embodiments, an interaction pair comprises any two chemical moieties that facilitate interaction of an associated non-luminescent pair. An interaction pair may consist of, for example: two complementary nucleic acids, two polypeptides capable of dimerization (e.g., homodimer, heterodimer, etc.), a protein and ligand, protein and small molecule, an antibody and epitope, a reactive pair of small molecules, etc. Any suitable pair of interacting molecules may find use as an interaction pair.

In some embodiments, an interaction pair comprises two molecules of interest (e.g., proteins of interest) or target molecules. In some embodiments, compositions and methods herein provide useful assays (e.g., in vitro, in vivo, in situ, whole animal, etc.) for studying the interactions between a pair of target molecules.

In certain embodiments, a pair off interaction elements, each attached to one of the non-luminescent elements, interact with each other and thereby facilitate formation of the bioluminescent complex. In some embodiments, the present of a ligand, substrate, co-factor or addition interaction element (e.g., not attached to non-luminescent element) is necessary to induce the interaction between the interaction elements and facilitate bioluminescent complex formation. In some embodiments, detecting a signal from the bioluminescent complex indicates the presence of the ligand, substrate, co-factor or addition interaction element or conditions that allow for interaction with the interaction elements.

In some embodiments, a pair off interaction elements, and a pair of non-luminescent elements are all present in a single amino acid chain (e.g., (interaction element 1)-NLpep-(interaction element 2)-NLpoly, NLpoly-(interaction element 1)-NLpep-(interaction element 2), NLpoly-(interaction element 1)-(interaction element 2)-NLpep, etc.). In some embodiments in which a pair off interaction elements, and a pair of non-luminescent elements are all present in a single amino acid chain, a ligand, substrate, co-factor or addition interaction element is required for the interaction pair to form an interaction complex and facilitate formation of the bioluminescent complex.

In certain embodiments, an interaction element and a non-luminescent element are attached, fused, linked, connected, etc. In typical embodiments, a first non-luminescent element and a first interaction element are attached to each other, and a second non-luminescent element and a second interaction element are attached to each other. Attachment of signal and interaction elements may be achieved by any suitable mechanism, chemistry, linker, etc. The interaction and non-luminescent elements are typically attached through covalent connection, but non-covalent linking of the two elements is also provided. In some embodiments, the signal and interaction elements are directly connected and, in other embodiments, they are connected by a linker.

In some embodiments, in which the interaction element is a peptide or polypeptide, the signal and interaction elements are contained within a single amino acid chain. In some embodiments, a single amino acid chain comprises, consists of, or consists essentially of a non-luminescent element and interaction element. In some embodiments, a single amino acid chain comprises, consists of, or consists essentially of a non-luminescent element, an interaction element, and optionally one or more an N-terminal sequence, a C-terminal sequence, regulatory elements (e.g., promoter, translational start site, etc.), and a linker sequence. In some embodiments, the signal and interaction elements are contained within a fusion polypeptide. The signal and interaction elements (and any other amino acid segments to be included in the fusion) may be expressed separately; however, in other embodiments, a fusion protein is expressed that comprises or consist of both the interaction and signal sequences.

In some embodiments, a first fusion protein comprising a first non-luminescent element and first interaction element as well as a second fusion protein comprising a second non-luminescent element and second interaction element are expressed within the same cells. In such embodiments, the first and second fusion proteins are purified and/or isolated from the cells, or the interaction of the fusion proteins is assayed within the cells. In other embodiments, first and second fusion proteins are expressed in separate cells and combined (e.g., following purification and/or isolation) for signal detection. In some embodiments, one or more fusion proteins are expressed in cell lysate (e.g., rabbit reticulocyte lysate) or in a cell-free system.

In certain embodiments, nucleic acids, DNA, RNA, vectors, etc. are provided that encode peptide, polypeptides, fusion polypeptide, fusion proteins, etc. of the present invention. Such nucleic acids and vectors may be used for expression, transformation, transfection, injection, etc.

In some embodiments, a non-luminescent element and interaction element are connected by a linker. In some embodiments, a linker connects the signal and interaction elements while providing a desired amount of space/distance between the elements. In some embodiments, a linker allows both the signal and interaction elements to form their respective pairs (e.g., non-luminescent pair and interaction pair) simultaneously. In some embodiments, a linker assists the interaction element in facilitating the formation of a non-luminescent pair interaction. In some embodiments, when an interaction pair is formed, the linkers that connect each non-luminescent element to their respective interaction elements position the non-luminescent elements at the proper distance and conformation to form a bioluminescent complex. In some embodiments, an interaction element and non-luminescent element are held in close proximity (e.g., <4 monomer units) by a linker. In some embodiments, a linker provides a desired amount of distance (e.g., 1, 2, 3, 4, 5, 6 . . . 10 . . . 20, or more monomer units) between signal and interaction elements (e.g., to prevent undesirable interactions between signal and interaction elements, for steric considerations, to allow proper orientation of non-luminescent element upon formation of interaction complex, to allow propagation of a complex-formation from interaction complex to non-luminescent elements, etc.). In certain embodiments, a linker provides appropriate attachment chemistry between the signal and interaction elements. A linker may also improve the synthetic process of making the signal and interaction element (e.g., allowing them to be synthesized as a single unit, allowing post synthesis connection of the two elements, etc.).

In some embodiments, a linker is any suitable chemical moiety capable of linking, connecting, or tethering a non-luminescent element to an interaction element. In some embodiments, a linker is a polymer of one or more repeating or non-repeating monomer units (e.g., nucleic acid, amino acid, carbon-containing polymer, carbon chain, etc.). When a non-luminescent element and interaction element are part of a fusion protein, a linker (when present) is typically an amino acid chain. When a non-luminescent element and interaction element are tethered together after the expression of the individual elements, a linker may comprise any chemical moiety with functional (or reactive) groups at either end that are reactive with functional groups on the signal and interaction elements, respectively. Any suitable moiety capable of tethering the signal and interaction elements may find use as a linker.

A wide variety of linkers may be used. In some embodiments, the linker is a single covalent bond. In some embodiments, the linker comprises a linear or branched, cyclic or heterocyclic, saturated or unsaturated, structure having 1-20 nonhydrogen atoms (e.g., C, N, P, O and S) and is composed of any combination of alkyl, ether, thioether, imine, carboxylic, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds. In some embodiments, linkers are longer than 20 nonhydrogen atoms (e.g. 21 non-hydrogen atoms, 25 non-hydrogen atoms, 30 non-hydrogen atoms, 40 non-hydrogen atoms, 50 non-hydrogen atoms, 100 non-hydrogen atoms, etc.) In some embodiments, the linker comprises 1-50 non-hydrogen atoms (in addition to hydrogen atoms) selected from the group of C, N, P, O and S (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 non-hydrogen atoms).

The present invention is not limited by the types of linkers available. The signal and interaction elements are linked, either directly (e.g. linker consists of a single covalent bond) or linked via a suitable linker. The present invention is not limited to any particular linker group. A variety of linker groups are contemplated, and suitable linkers could comprise, but are not limited to, alkyl groups, methylene carbon chains, ether, polyether, alkyl amide linker, a peptide linker, a modified peptide linker, a Poly(ethylene glycol) (PEG) linker, a streptavidin-biotin or avidin-biotin linker, polyaminoacids (e.g. polylysine), functionalised PEG, polysaccharides, glycosaminoglycans, dendritic polymers (WO93/06868 and by Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990), herein incorporated by reference in their entireties), PEG-chelant polymers (W94/08629, WO94/09056 and WO96/26754, herein incorporated by reference in their entireties), oligonucleotide linker, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfide, or a combination thereof. In some embodiments, the linker is cleavable (e.g., enzymatically (e.g., TEV protease site), chemically, photoinduced, etc.

In some embodiments, substantially non-luminescent peptides and polypeptides are provided with less than 100% sequence identity and/or similarity to any portion of an existing luciferase (e.g., a firefly luciferase, a Renilla luciferase, an Oplophorus luciferase, enhanced Oplophorus luciferases as described in U.S. Pat. App. 2010/0281552 and U.S. Pat. App. 2012/0174242, herein incorporated by reference in their entireties). Certain embodiments of the present invention involve the formation of bioluminescent complexes of non-luminescent peptides and polypeptides with less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with all or a portion (e.g., 8 or more amino acids, less than about 25 amino acids for peptides) of SEQ ID NO: 2157 (e.g., complete NANOLUC sequence). In some embodiments, non-luminescent peptides and polypeptides are provided with less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence similarity with a portion (e.g., 8 or more amino acids, less than about 25 amino acids for peptides) of SEQ ID NO: 2157 (e.g., peptides and polypeptides that interact to form bioluminescent complexes). Non-luminescent peptides are provided that have less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity and/or similarity with about a 25 amino acid or less portion of SEQ ID NO: 2157, wherein such peptides form a bioluminescent complex when combined under appropriate conditions (e.g., stabilized by an interaction pair) with a polypeptide having less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity and/or similarity with another portion SEQ ID NO: 2157. Similarly, non-luminescent polypeptides are provided that have less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity or similarity with a portion of SEQ ID NO: 2157, wherein such polypeptides form a bioluminescent complex when combined under appropriate conditions (e.g., stabilized by an interaction pair) with a peptide having less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity and/or similarity with another portion SEQ ID NO: 2157. In some embodiments, non-luminescent peptides with less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity or similarity with SEQ ID NO: 2 are provided. In some embodiments, non-luminescent polypeptides with less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity or similarity with SEQ ID NO: 440 are provided.

In some embodiments, non-luminescent peptides that find use in embodiments of the present invention include peptides with one or more amino acid substitutions, deletions, or additions from GVTGWRLCKRISA (SEQ ID NO: 236). In some embodiments, the present invention provides peptides comprising one or more of amino acid sequences of Table 1, and/or nucleic acids comprising the nucleic acid sequences of Table 1 (which code for the peptide sequences of Table 1).

TABLE 1 Peptide sequences SEQ ID NO. PEPTIDE NO. POLYMER SEQUENCE 3 NLpep2 (w/ Met) N.A. ATGGACGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCG 4 NLpep2 (w/ Met) A.A. MDVTGWRLCERILA 5 NLpep3 (w/ Met) N.A. ATGGGAGTGACCGCCTGGCGGCTGTGCGAACGCATTCTGGCG 6 NLpep3 (w/ Met) A.A. MGVTAWRLCERILA 7 NLpep4 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTCTGGCG 8 NLpep4 (w/ Met) A.A. MGVTGWRLCKRILA 9 NLpep5 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTAGCGCG 10 NLpep5 (w/ Met) A.A. MGVTGWRLCERISA 11 NLpep6 (w/ Met) N.A. ATGGACGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 12 NLpep6 (w/ Met) A.A. MDVTGWRLCKRISA 13 NLpep7 (w/ Met) N.A. ATGGACGTGACCGGCTGGCGGCTGTGCAAGCGCATTCTGGCG 14 NLpep7 (w/ Met) A.A. MDVTGWRLCKRILA 15 NLpep8 (w/ Met) N.A. ATGGACGTGACCGGCTGGCGGCTGTGCGAACGCATTAGCGCG 16 NLpep8 (w/ Met) A.A. MDVTGWRLCERISA 17 NLpep9 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 18 NLpep9 (w/ Met) A.A. MGVTGWRLCKRISA 19 NLpep10 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGAACGAACGCATTCTGGCG 20 NLpep10 (w/ Met) A.A. MGVTGWRLNERILA 21 NLpep11 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGCAGGAACGCATTCTGGCG 22 NLpep11 (w/ Met) A.A. MGVTGWRLQERILA 23 NLpep12 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGAAGAAGCGCCGGAGCCGG 24 NLpep12 (w/ Met) A.A. MGVTGWRLKKRRSR 25 NLpep13 (w/ Met) N.A. ATGAACGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 26 NLpep13 (w/ Met) A.A. MNVTGWRLCKRISA 27 NLpep14 (w/ Met) N.A. ATGAGCGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 28 NLpep14 (w/ Met) A.A. MSVTGWRLCKRISA 29 NLpep15 (w/ Met) N.A. ATGGAGGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 30 NLpep15 (w/ Met) A.A. MEVTGWRLCKRISA 31 NLpep16 (w/ Met) N.A. ATGGGCGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 32 NLpep16 (w/ Met) A.A. MGVTGWRLCKRISA 33 NLpep17 (w/ Met) N.A. ATGGGACACACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 34 NLpep17 (w/ Met) A.A. MGITGWRLCKRISA 35 NLpep18 (w/ Met) N.A. ATGGGAGCCACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 36 NLpep18 (w/ Met) A.A. MGATGWRLCKRISA 37 NLpep19 (w/ Met) N.A. ATGGGAAAGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 38 NLpep19 (w/ Met) A.A. MGKTGWRLCKRISA 39 NLpep20 (w/ Met) N.A. ATGGGACAGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 40 NLpep20 (w/ Met) A.A. MGQTGWRLCKRISA 41 NLpep21 (w/ Met) N.A. ATGGGAAGCACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 42 NLpep21 (w/ Met) A.A. MGSTGWRLCKRISA 43 NLpep22 (w/ Met) N.A. ATGGGAGTGGTGGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 44 NLpep22 (w/ Met) A.A. MGVVGWRLCKRISA 45 NLpep23 (w/ Met) N.A. ATGGGAGTGAAGGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 46 NLpep23 (w/ Met) A.A. MGVKGWRLCKRISA 47 NLpep24 (w/ Met) N.A. ATGGGAGTGCAGGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 48 NLpep24 (w/ Met) A.A. MGVQGWRLCKRISA 49 NLpep25 (w/ Met) N.A. ATGGGAGTGACCGGCACCCGGCTGTGCAAGCGCATTAGCGCG 50 NLpep25 (w/ Met) A.A. MGVTGTRLCKRISA 51 NLpep26 (w/ Met) N.A. ATGGGAGTGACCGGCAAGCGGCTGTGCAAGCGCATTAGCGCG 52 NLpep26 (w/ Met) A.A. MGVTGKRLCKRISA 53 NLpep27 (w/ Met) N.A. ATGGGAGTGACCGGCGTGCGGCTGTGCAAGCGCATTAGCGCG 54 NLpep27 (w/ Met) A.A. MGVTGVRLCKRISA 55 NLpep28 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCACTGCAAGCGCATTAGCGCG 56 NLpep28 (w/ Met) A.A. MGVTGWRICKRISA 57 NLpep29 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGGTGTGCAAGCGCATTAGCGCG 58 NLpep29 (w/ Met) A.A. MGVTGWRVCKRISA 59 NLpep30 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGACCTGCAAGCGCATTAGCGCG 60 NLpep30 (w/ Met) A.A. MGVTGWRTCKRISA 61 NLpep31 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGTACTGCAAGCGCATTAGCGCG 62 NLpep31 (w/ Met) A.A. MGVTGWRYCKRISA 63 NLpep32 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGAAGTGCAAGCGCATTAGCGCG 64 NLpep32 (w/ Met) A.A. MGVTGWRKCKRISA 65 NLpep33 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGAACAAGCGCATTAGCGCG 66 NLpep33 (w/ Met) A.A. MGVTGWRLNKRISA 67 NLpep34 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGACCAAGCGCATTAGCGCG 68 NLpep34 (w/ Met) A.A. MGVTGWRLTKRISA 69 NLpep35 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGAAGATTAGCGCG 70 NLpep35 (w/ Met) A.A. MGVTGWRLCKKISA 71 NLpep36 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGAACATTAGCGCG 72 NLpep36 (w/ Met) A.A. MGVTGWRLCKNISA 73 NLpep37 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCGTGAGCGCG 74 NLpep37 (w/ Met) A.A. MGVTGWRLCKRVSA 75 NLpep38 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCCAGAGCGCG 76 NLpep38 (w/ Met) A.A. MGVTGWRLCKRQSA 77 NLpep39 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCGAGAGCGCG 78 NLpep39 (w/ Met) A.A. MGVTGWRLCKRESA 79 NLpep40 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCCGGAGCGCG 80 NLpep40 (w/ Met) A.A. MGVTGWRLCKRRSA 81 NLpep41 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCTTCAGCGCG 82 NLpep41 (w/ Met) A.A. MGVTGWRLCKRFSA 83 NLpep42 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCAAC 84 NLpep42 (w/ Met) A.A. MGVTGWRLCKRISN 85 NLpep43 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCACC 86 NLpep43 (w/ Met) A.A. MGVTGWRLCKRIST 87 NLpep44 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCCGG 88 NLpep44 (w/ Met) A.A. MGVTGWRLCKRISR 89 NLpep45 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCCTG 90 NLpep45 (w/ Met) A.A. MGVTGWRLCKRISL 91 NLpep46 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGAG 92 NLpep46 (w/ Met) A.A. MGVTGWRLCKRISE 93 NLpep47 (w/ Met) N.A. ATGGGAGTGACCGGCTTCCGGCTGTGCAAGCGCATTAGCGCG 94 NLpep47 (w/ Met) A.A. MGVTGFRLCKRISA 95 NLpep48 (w/ Met) N.A. ATGGGAGTGACCGGCTACCGGCTGTGCAAGCGCATTAGCGCG 96 NLpep48 (w/ Met) A.A. MGVTGYRLCKRISA 97 NLpep49 (w/ Met)  N.A. ATGGGAGTGACCGGCGAGCGGCTGTGCAAGCGCATTAGCGCG 98 NLpep49 (w/ Met)  A.A. MGVTGERLCKRISA 99 NLpep50 (w/ Met) N.A. ATGCAGGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 100 NLpep50 (w/ Met) A.A. MQVTGWRLCKRISA 101 NLpep51 (w/ Met) N.A. ATGACCGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 102 NLpep51 (w/ Met) A.A. MTVTGWRLCKRISA 103 NLpep52 (w/ Met) N.A. ATGGGAGTGGAGGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 104 NLpep52 (w/ Met) A.A. MGVEGWRLCKRISA 105 NLpep53 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCGCG 106 NLpep53 (w/ Met) A.A. MGVTGWRLFKRISA 107 NLpep54 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTACAAGCGCATTAGCGCG 108 NLpep54 (w/ Met) A.A. MGVTGWRLYKRISA 109 NLpep55 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGAGCAAGCGCATTAGCGCG 110 NLpep55 (w/ Met) A.A. MGVTGWRLSKRISA 111 NLpep56 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGGGCAAGCGCATTAGCGCG 112 NLpep56 (w/ Met) A.A. MGVTGWRLHKRISA 113 NLpep57 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGATGAAGCGCATTAGCGCG 114 NLpep57 (w/ Met) A.A. MGVTGWRLMKRISA 115 NLpep58 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGGCCAAGCGCATTAGCGCG 116 NLpep58 (w/ Met) A.A. MGVTGWRLAKRISA 117 NLpep59 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGCAGAAGCGCATTAGCGCG 118 NLpep59 (w/ Met) A.A. MGVTGWRLQKRISA 119 NLpep60 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGCTGAAGCGCATTAGCGCG 120 NLpep60 (w/ Met) A.A. MGVTGWRLLKRISA 121 NLpep61 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGAAGAAGCGCATTAGCGCG 122 NLpep61 (w/ Met) A.A. MGVTGWRLKKRISA 123 NLpep62 (w/ Met) N.A. ATGAACCACACCGGCTGGCGGCTGAACAAGAAGGTGAGCAAC 124 NLpep62 (w/ Met) A.A. MNITGWRLNKKVSN 125 NLpep63 (w/ Met) N.A. ATGAACCACACCGGCTACCGGCTGAACAAGAAGGTGAGCAAC 126 NLpep63 (w/ Met) A.A. MNITGYRLNKKVSN 127 NLpep64 (w/ Met) N.A. ATGTGCGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCGCG 128 NLpep64 (w/ Met) A.A. MCVTGWRLFKRISA 129 NLpep65 (w/ Met) N.A. ATGCCCGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCGCG 130 NLpep65 (w/ Met) A.A. MPVTGWRLFKRISA 131 NLpep66 (w/ Met) N.A. ATGAACCACACCGGCTACCGGCTGTTCAAGAAGGTGAGCAAC 132 NLpep66 (w/ Met) A.A. MNITGYRLFKKVSN 133 NLpep67 (w/ Met) N.A. ATGAACGTGACCGGCTACCGGCTGTTCAAGAAGGTGAGCAAC 134 NLpep67 (w/ Met) A.A. MNVTGYRLFKKVSN 135 NLpep68 (w/ Met) N.A. ATGAACGTGACCGGCTGGCGGCTGTTCAAGAAGGTGAGCAAC 136 NLpep68 (w/ Met) A.A. MNVTGWRLFKKVSN 137 NLpep69 (w/ Met) N.A. ATGAACGTGACCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 138 NLpep69 (w/ Met) A.A. MNVTGWRLFKKISN 139 NLpep70 (w/ Met) N.A. ATGAACGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCAAC 140 NLpep70 (w/ Met) A.A. MNVTGWRLFKRISN 141 NLpep71 (w/ Met) N.A. ATGGGAGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCAAC 142 NLpep71 (w/ Met) A.A. MGVTGWRLFKRISN 143 NLpep72 (w/ Met) N.A. ATGAACGTGACCGGCTGGCGGCTGTTCGAACGCATTAGCAAC 144 NLpep72 (w/ Met) A.A. MNVTGWRLFERISN 145 NLpep73 (w/ Met) N.A. ATGAACGTGACCGGCTGGCGGCTGTTCAAGCGCATTCTGAAC 146 NLpep73 (w/ Met) A.A. MNVTGWRLFKRILN 147 NLpep74 (w/ Met) N.A. ATGAACGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCGCG 148 NLpep74 (w/ Met) A.A. MNVTGWRLFKRISA 149 NLpep75 (w/ Met) N.A. ATGAACGTGACCGGCTGGCGGCTGTTCGAAAAGATTAGCAAC 150 NLpep75 (w/ Met) A.A. MNVTGWRLFEKISN 151 NLpep76 (w/ Met) N.A. ATGAACGTGAGCGGCTGGCGGCTGTTCGAAAAGATTAGCAAC 152 NLpep76 (w/ Met) A.A. MNVSGWRLFEKISN 153 NLpep77 (w/ Met) N.A. ATG-GTGACCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 154 NLpep77 (w/ Met) A.A. M-VTGWRLFKKISN 155 NLpep78 (w/ Met) N.A. ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 156 NLpep78 (w/ Met) A.A. MNVSGWRLFKKISN 157 NLpep79 (w/ Met) N.A. ATGAACGTGACCGGCTACCGGCTGTTCAAGAAGATTAGCAAC 158 NLpep79 (w/ Met) A.A. MNVTGYRLFKKISN 159 NLpep80 (w/ Met)  N.A. ATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 160 NLpep80 (w/ Met)  A.A. MVSGWRLFKKISN 161 NLpep81 (w/ Met) N.A. ATGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 440 NLpep81 (w/ Met) A.A. MSGWRLFKKISN 163 NLpep82 (w/ Met) N.A. ATGGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 164 NLpep82 (w/ Met) A.A. MGWRLFKKISN 165 NLpep83 (w/ Met) N.A. ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC 166 NLpep83 (w/ Met) A.A. MNVSGWRLFKKIS 167 NLpep84 (w/ Met) N.A. ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAGATT 168 NLpep84 (w/ Met) A.A. MNVSGWRLFKKI 169 NLpep85 (w/ Met) N.A. ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAG 170 NLpep85 (w/ Met) A.A. MNVSGWRLFKK 171 NLpep86 (w/ Met) N.A. ATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC 172 NLpep86 (w/ Met) A.A. MVSGWRLFKKIS 173 NLpep87 (w/ Met) N.A. ATGAGCGGCTGGCGGCTGTTCAAGAAGATT 174 NLpep87 (w/ Met) A.A. MSGWRLFKKI 175 NLpep88 (w/ Met) N.A. ATGAACGTGAGCGGCTGGGGCCTGTTCAAGAAGATTAGCAAC 176 NLpep88 (w/ Met) A.A. MNVSGWGLFKKISN 177 NLpep89 (w/ Met) N.A. ATGCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 178 NLpep89 (w/ Met) A.A. MPVSGWRLFKKISN 179 NLpep90 (w/ Met) N.A. ATGAACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 180 NLpep90 (w/ Met) A.A. MNPVSGWRLFKKISN 181 NLpep91 (w/ Met) N.A. ATGATCAACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 182 NLpep91 (w/ Met) A.A. MINPVSGWRLFKKISN 183 NLpep92 (w/ Met) N.A. ATGACCATCAACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 184 NLpep92 (w/ Met) A.A. MTINPVSGWRLFKKISN 185 NLpep93 (w/ Met) N.A. ATGGTGACCATCAACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 186 NLpep93 (w/ Met) A.A. MVTINPVSGWRLFKKISN 187 NLpep94 (w/ Met) N.A. ATGCGGGTGACCATCAACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 188 NLpep94 (w/ Met) A.A. MRVTINPVSGWRLFKKISN 189 NLpep95 (w/ Met) N.A. ATGAGCGGCTGGCGGCTGCTGAAGAAGATT 190 NLpep95 (w/ Met) A.A. MSGWRLLKKI 191 NLpep96 (w/ Met) N.A. ATGACCGGCTACCGGCTGCTGAAGAAGATT 192 NLpep96 (w/ Met) A.A. MTGYRLLKKI 193 NLpep97 (w/ Met) N.A. ATGAGCGGCTGGCGGCTGTTCAAGAAG 194 NLpep97 (w/ Met) A.A. MSGWRLFKK 195 NLpep98 (w/ Met) N.A. ATGGTGACCGGCTACCGGCTGTTCAAGAAGATTAGC 196 NLpep98 (w/ Met) A.A. MVTGYRLFKKIS 197 NLpep99 (w/ Met) N.A. ATGGTGACCGGCTACCGGCTGTTCGAGAAGATTAGC 198 NLpep99 (w/ Met) A.A. MVTGYRLFEKIS 199 NLpep100 (w/ Met) N.A. ATGGTGACCGGCTACCGGCTGTTCGAGCAGATTAGC 200 NLpep100 (w/ Met) A.A. MVTGYRLFEQIS 201 NLpep101 (w/ Met) N.A. ATGGTGACCGGCTACCGGCTGTTCGAGAAGGAGAGC 202 NLpep101 (w/ Met) A.A. MVTGYRLFEKES 203 NLpep102 (w/ Met) N.A. ATGGTGACCGGCTACCGGCTGTTCGAGCAGGAGAGC 204 NLpep102 (w/ Met) A.A. MVTGYRLFEQES 205 NLpep103 (w/ Met) N.A. ATGGTGACCGGCTACCGGCTGTTCGAGCAGGAGCTG 206 NLpep103 (w/ Met) A.A. MVTGYRLFEQEL 207 NLpep104 (w/ Met) N.A. ATGGTGGAGGGCTACCGGCTGTTCGAGAAGATTAGC 208 NLpep104 (w/ Met) A.A. MVEGYRLFEKIS 209 NLpep105 (w/ Met) N.A. ATGGTGGAGGGCTACCGGCTGTTCGAGCAGATTAGC 210 NLpep105 (w/ Met) A.A. MVEGYRLFEQIS 211 NLpep106 (w/ Met) N.A. ATGGTGGAGGGCTACCGGCTGTTCGAGAAGGAGAGC 212 NLpep106 (w/ Met) A.A. MVEGYRLFEKES 213 NLpep107 (w/ Met) N.A. ATGGTGGAGGGCTACCGGCTGTTCGAGCAGGAGAGC 214 NLpep107 (w/ Met) A.A. MVEGYRLFEQES 215 NLpep108 (w/ Met) N.A. ATGGTGGAGGGCTACCGGCTGTTCGAGCAGGAGCTG 216 NLpep108 (w/ Met) A.A. MVEGYRLFEQEL 217 NLpep109 (w/ Met) N.A. ATGATTAGCGGCTGGCGGCTGATGAAGAACATTAGC 218 NLpep109 (w/ Met) A.A. MISGWRLMKNIS 219 NLpep110 (w/ Met) N.A. ATGGTGGAGGGCTACCGGCTGTTCAAGAAGATTAGC 220 NLpep110 (w/ Met) A.A. MVEGYRLFKKIS 221 NLpep2 (w/o Met)  N.A. GACGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCG 222 NLpep2 (w/o Met)  A.A. DVTGWRLCERILA 223 NLpep3 (w/o Met)  N.A. GGAGTGACCGCCTGGCGGCTGTGCGAACGCATTCTGGCG 224 NLpep3 (w/o Met)  A.A. GVTAWRLCERILA 225 NLpep4 (w/o Met)  N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTCTGGCG 226 NLpep4 (w/o Met)  A.A. GVTGWRLCKRILA 227 NLpep5 (w/o Met)  N.A. GGAGTGACCGGCTGGCGGCTGTGCGAACGCATTAGCGCG 228 NLpep5 (w/o Met)  A.A. GVTGWRLCERISA 229 NLpep6 (w/o Met)  N.A. GACGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 230 NLpep6 (w/o Met)  A.A. DVTGWRLCKRISA 231 NLpep7 (w/o Met)  N.A. GACGTGACCGGCTGGCGGCTGTGCAAGCGCATTCTGGCG 232 NLpep7 (w/o Met)  A.A. DVTGWRLCKRILA 233 NLpep8 (w/o Met)  N.A. GACGTGACCGGCTGGCGGCTGTGCGAACGCATTAGCGCG 234 NLpep8 (w/o Met)  A.A. DVTGWRLCERISA 235 NLpep9 (w/o Met)  N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 236 NLpep9 (w/o Met)  A.A. GVTGWRLCKRISA 237 NLpep10 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGAACGAACGCATTCTGGCG 238 NLpep10 (w/o Met) A.A. GVTGWRLNERILA 239 NLpep11 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGCAGGAACGCATTCTGGCG 240 NLpep11 (w/o Met) A.A. GVTGWRLQERILA 241 NLpep12 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGAAGAAGCGCCGGAGCCGG 242 NLpep12 (w/o Met) A.A. GVTGWRLKKRRSR 243 NLpep13 (w/o Met) N.A. AACGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 244 NLpep13 (w/o Met) A.A. NVTGWRLCKRISA 245 NLpep14 (w/o Met) N.A. AGCGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 246 NLpep14 (w/o Met) A.A. SVTGWRLCKRISA 247 NLpep15 (w/o Met) N.A. GAGGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 248 NLpep15 (w/o Met) A.A. EVTGWRLCKRISA 249 NLpep16 (w/o Met) N.A. GGCGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 250 NLpep16 (w/o Met) A.A. HVTGWRLCKRISA 251 NLpep17 (w/o Met) N.A. GGACACACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 252 NLpep17 (w/o Met) A.A. GITGWRLCKRISA 253 NLpep18 (w/o Met) N.A. GGAGCCACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 254 NLpep18 (w/o Met) A.A. GATGWRLCKRISA 255 NLpep19 (w/o Met) N.A. GGAAAGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 256 NLpep19 (w/o Met) A.A. GKTGWRLCKRISA 257 NLpep20 (w/o Met) N.A. GGACAGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 258 NLpep20 (w/o Met) A.A. GQTGWRLCKRISA 259 NLpep21 (w/o Met) N.A. GGAAGCACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 260 NLpep21 (w/o Met) A.A. GSTGWRLCKRISA 261 NLpep22 (w/o Met) N.A. GGAGTGGTGGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 262 NLpep22 (w/o Met) A.A. GVVGWRLCKRISA 263 NLpep23 (w/o Met) N.A. GGAGTGAAGGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 264 NLpep23 (w/o Met) A.A. GVKGWRLCKRISA 265 NLpep24 (w/o Met) N.A. GGAGTGCAGGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 266 NLpep24 (w/o Met) A.A. GVQGWRLCKRISA 267 NLpep25 (w/o Met) N.A. GGAGTGACCGGCACCCGGCTGTGCAAGCGCATTAGCGCG 268 NLpep25 (w/o Met) A.A. GVTGTRLCKRISA 269 NLpep26 (w/o Met) N.A. GGAGTGACCGGCAAGCGGCTGTGCAAGCGCATTAGCGCG 270 NLpep26 (w/o Met) A.A. GVTGKRLCKRISA 271 NLpep27 (w/o Met) N.A. GGAGTGACCGGCGTGCGGCTGTGCAAGCGCATTAGCGCG 272 NLpep27 (w/o Met) A.A. GVTGVRLCKRISA 273 NLpep28 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCACTGCAAGCGCATTAGCGCG 274 NLpep28 (w/o Met) A.A. GVTGWRICKRISA 275 NLpep29 (w/o Met) N.A. GGAGTGACCGGCTGGCGGGTGTGCAAGCGCATTAGCGCG 276 NLpep29 (w/o Met) A.A. GVTGWRVCKRISA 277 NLpep30 (w/o Met) N.A. GGAGTGACCGGCTGGCGGACCTGCAAGCGCATTAGCGCG 278 NLpep30 (w/o Met) A.A. GVTGWRTCKRISA 279 NLpep31 (w/o Met) N.A. GGAGTGACCGGCTGGCGGTACTGCAAGCGCATTAGCGCG 280 NLpep31 (w/o Met) A.A. GVTGWRYCKRISA 281 NLpep32 (w/o Met) N.A. GGAGTGACCGGCTGGCGGAAGTGCAAGCGCATTAGCGCG 282 NLpep32 (w/o Met) A.A. GVTGWRKCKRISA 283 NLpep33 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGAACAAGCGCATTAGCGCG 284 NLpep33 (w/o Met) A.A. GVTGWRLNKRISA 285 NLpep34 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGACCAAGCGCATTAGCGCG 286 NLpep34 (w/o Met) A.A. GVTGWRLTKRISA 287 NLpep35 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGAAGATTAGCGCG 288 NLpep35 (w/o Met) A.A. GVTGWRLCKKISA 289 NLpep36 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGAACATTAGCGCG 290 NLpep36 (w/o Met) A.A. GVTGWRLCKNISA 291 NLpep37 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCGTGAGCGCG 292 NLpep37 (w/o Met) A.A. GVTGWRLCKRVSA 293 NLpep38 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCCAGAGCGCG 294 NLpep38 (w/o Met) A.A. GVTGWRLCKRQSA 295 NLpep39 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCGAGAGCGCG 296 NLpep39 (w/o Met) A.A. GVTGWRLCKRESA 297 NLpep40 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCCGGAGCGCG 298 NLpep40 (w/o Met) A.A. GVTGWRLCKRRSA 299 NLpep41 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCTTCAGCGCG 300 NLpep41 (w/o Met) A.A. GVTGWRLCKRFSA 301 NLpep42 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCAAC 302 NLpep42 (w/o Met) A.A. GVTGWRLCKRISN 303 NLpep43 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCACC 304 NLpep43 (w/o Met) A.A. GVTGWRLCKRIST 305 NLpep44 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCCGG 306 NLpep44 (w/o Met) A.A. GVTGWRLCKRISR 307 NLpep45 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCCTG 308 NLpep45 (w/o Met) A.A. GVTGWRLCKRISL 309 NLpep46 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGAG 310 NLpep46 (w/o Met) A.A. GVTGWRLCKRISE 311 NLpep47 (w/o Met) N.A. GGAGTGACCGGCTTCCGGCTGTGCAAGCGCATTAGCGCG 312 NLpep47 (w/o Met) A.A. GVTGFRLCKRISA 313 NLpep48 (w/o Met) N.A. GGAGTGACCGGCTACCGGCTGTGCAAGCGCATTAGCGCG 314 NLpep48 (w/o Met) A.A. GVTGYRLCKRISA 315 NLpep49 (w/o Met)  N.A. GGAGTGACCGGCGAGCGGCTGTGCAAGCGCATTAGCGCG 316 NLpep49 (w/o Met)  A.A. GVTGERLCKRISA 317 NLpep50 (w/o Met) N.A. CAGGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 318 NLpep50 (w/o Met) A.A. QVTGWRLCKRISA 319 NLpep51 (w/o Met) N.A. ACCGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 320 NLpep51 (w/o Met) A.A. TVTGWRLCKRISA 321 NLpep52 (w/o Met) N.A. GGAGTGGAGGGCTGGCGGCTGTGCAAGCGCATTAGCGCG 322 NLpep52 (w/o Met) A.A. GVEGWRLCKRISA 323 NLpep53 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCGCG 324 NLpep53 (w/o Met) A.A. GVTGWRLFKRISA 325 NLpep54 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTACAAGCGCATTAGCGCG 326 NLpep54 (w/o Met) A.A. GVTGWRLYKRISA 327 NLpep55 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGAGCAAGCGCATTAGCGCG 328 NLpep55 (w/o Met) A.A. GVTGWRLSKRISA 329 NLpep56 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGGGCAAGCGCATTAGCGCG 330 NLpep56 (w/o Met) A.A. GVTGWRLHKRISA 331 NLpep57 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGATGAAGCGCATTAGCGCG 332 NLpep57 (w/o Met) A.A. GVTGWRLMKRISA 333 NLpep58 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGGCCAAGCGCATTAGCGCG 334 NLpep58 (w/o Met) A.A. GVTGWRLAKRISA 335 NLpep59 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGCAGAAGCGCATTAGCGCG 336 NLpep59 (w/o Met) A.A. GVTGWRLQKRISA 337 NLpep60 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGCTGAAGCGCATTAGCGCG 338 NLpep60 (w/o Met) A.A. GVTGWRLLKRISA 339 NLpep61 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGAAGAAGCGCATTAGCGCG 340 NLpep61 (w/o Met) A.A. GVTGWRLKKRISA 341 NLpep62 (w/o Met) N.A. AACCACACCGGCTGGCGGCTGAACAAGAAGGTGAGCAAC 342 NLpep62 (w/o Met) A.A. NITGWRLNKKVSN 343 NLpep63 (w/o Met) N.A. AACCACACCGGCTACCGGCTGAACAAGAAGGTGAGCAAC 344 NLpep63 (w/o Met) A.A. NITGYRLNKKVSN 345 NLpep64 (w/o Met) N.A. TGCGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCGCG 346 NLpep64 (w/o Met) A.A. CVTGWRLFKRISA 347 NLpep65 (w/o Met) N.A. CCCGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCGCG 348 NLpep65 (w/o Met) A.A. PVTGWRLFKRISA 349 NLpep66 (w/o Met) N.A. AACCACACCGGCTACCGGCTGTTCAAGAAGGTGAGCAAC 350 NLpep66 (w/o Met) A.A. NITGYRLFKKVSN 351 NLpep67 (w/o Met) N.A. AACGTGACCGGCTACCGGCTGTTCAAGAAGGTGAGCAAC 352 NLpep67 (w/o Met) A.A. NVTGYRLFKKVSN 353 NLpep68 (w/o Met) N.A. AACGTGACCGGCTGGCGGCTGTTCAAGAAGGTGAGCAAC 354 NLpep68 (w/o Met) A.A. NVTGWRLFKKVSN 355 NLpep69 (w/o Met) N.A. AACGTGACCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 356 NLpep69 (w/o Met) A.A. NVTGWRLFKKISN 357 NLpep70 (w/o Met) N.A. AACGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCAAC 358 NLpep70 (w/o Met) A.A. NVTGWRLFKRISN 359 NLpep71 (w/o Met) N.A. GGAGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCAAC 360 NLpep71 (w/o Met) A.A. GVTGWRLFKRISN 361 NLpep72 (w/o Met) N.A. AACGTGACCGGCTGGCGGCTGTTCGAACGCATTAGCAAC 362 NLpep72 (w/o Met) A.A. NVTGWRLFERISN 363 NLpep73 (w/o Met) N.A. AACGTGACCGGCTGGCGGCTGTTCAAGCGCATTCTGAAC 364 NLpep73 (w/o Met) A.A. NVTGWRLFKRILN 365 NLpep74 (w/o Met) N.A. AACGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCGCG 366 NLpep74 (w/o Met) A.A. NVTGWRLFKRISA 367 NLpep75 (w/o Met) N.A. AACGTGACCGGCTGGCGGCTGTTCGAAAAGATTAGCAAC 368 NLpep75 (w/o Met) A.A. NVTGWRLFEKISN 369 NLpep76 (w/o Met) N.A. AACGTGAGCGGCTGGCGGCTGTTCGAAAAGATTAGCAAC 370 NLpep76 (w/o Met) A.A. NVSGWRLFEKISN 371 NLpep77 (w/o Met) N.A. GTGACCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 372 NLpep77 (w/o Met) A.A. VTGWRLFKKISN 373 NLpep78 (w/o Met) N.A. AACGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 374 NLpep78 (w/o Met) A.A. NVSGWRLFKKISN 375 NLpep79 (w/o Met) N.A. AACGTGACCGGCTACCGGCTGTTCAAGAAGATTAGCAAC 376 NLpep79 (w/o Met) A.A. NVTGYRLFKKISN 377 NLpep80 (w/o Met)  N.A. GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 378 NLpep80 (w/o Met)  A.A. VSGWRLFKKISN 379 NLpep81 (w/o Met) N.A. AGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 380 NLpep81 (w/o Met) A.A. SGWRLFKKISN 381 NLpep82 (w/o Met) N.A. GGCTGGCGGCTGTTCAAGAAGATTAGCAAC 382 NLpep82 (w/o Met) A.A. GWRLFKKISN 383 NLpep83 (w/o Met) N.A. AACGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC 384 NLpep83 (w/o Met) A.A. NVSGWRLFKKIS 385 NLpep84 (w/o Met) N.A. AACGTGAGCGGCTGGCGGCTGTTCAAGAAGATT 386 NLpep84 (w/o Met) A.A. NVSGWRLFKKI 387 NLpep85 (w/o Met) N.A. AACGTGAGCGGCTGGCGGCTGTTCAAGAAG 388 NLpep85 (w/o Met) A.A. NVSGWRLFKK 389 NLpep86 (w/o Met) N.A. GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC 390 NLpep86 (w/o Met) A.A. VSGWRLFKKIS 391 NLpep87 (w/o Met) N.A. AGCGGCTGGCGGCTGTTCAAGAAGATT 392 NLpep87 (w/o Met) A.A. SGWRLFKKI 393 NLpep88 (w/o Met) N.A. AACGTGAGCGGCTGGGGCCTGTTCAAGAAGATTAGCAAC 394 NLpep88 (w/o Met) A.A. NVSGWGLFKKISN 395 NLpep89 (w/o Met) N.A. CCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 396 NLpep89 (w/o Met) A.A. PVSGWRLFKKISN 397 NLpep90 (w/o Met) N.A. AACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 398 NLpep90 (w/o Met) A.A. NPVSGWRLFKKISN 399 NLpep91 (w/o Met) N.A. ATCAACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 400 NLpep91 (w/o Met) A.A. INPVSGWRLFKKISN 401 NLpep92 (w/o Met) N.A. ACCATCAACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 402 NLpep92 (w/o Met) A.A. TINPVSGWRLFKKISN 403 NLpep93 (w/o Met) N.A. GTGACCATCAACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 404 NLpep93 (w/o Met) A.A. VTINPVSGWRLFKKISN 405 NLpep94 (w/o Met) N.A. CGGGTGACCATCAACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 406 NLpep94 (w/o Met) A.A. RVTINPVSGWRLFKKISN 407 NLpep95 (w/o Met) N.A. AGCGGCTGGCGGCTGCTGAAGAAGATT 408 NLpep95 (w/o Met) A.A. SGWRLLKKI 409 NLpep96 (w/o Met) N.A. ACCGGCTACCGGCTGCTGAAGAAGATT 410 NLpep96 (w/o Met) A.A. TGYRLLKKI 411 NLpep97 (w/o Met) N.A. AGCGGCTGGCGGCTGTTCAAGAAG 412 NLpep97 (w/o Met) A.A. SGWRLFKK 413 NLpep98 (w/o Met) N.A. GTGACCGGCTACCGGCTGTTCAAGAAGATTAGC 414 NLpep98 (w/o Met) A.A. VTGYRLFKKIS 415 NLpep99 (w/o Met) N.A. GTGACCGGCTACCGGCTGTTCGAGAAGATTAGC 416 NLpep99 (w/o Met) A.A. VTGYRLFEKIS 417 NLpep100 (w/o Met) N.A. GTGACCGGCTACCGGCTGTTCGAGCAGATTAGC 418 NLpep100 (w/o Met) A.A. VTGYRLFEQIS 419 NLpep101 (w/o Met) N.A. GTGACCGGCTACCGGCTGTTCGAGAAGGAGAGC 420 NLpep101 (w/o Met) A.A. VTGYRLFEKES 421 NLpep102 (w/o Met) N.A. GTGACCGGCTACCGGCTGTTCGAGCAGGAGAGC 422 NLpep102 (w/o Met) A.A. VTGYRLFEQES 423 NLpep103 (w/o Met) N.A. GTGACCGGCTACCGGCTGTTCGAGCAGGAGCTG 424 NLpep103 (w/o Met) A.A. VTGYRLFEQEL 425 NLpep104 (w/o Met) N.A. GTGGAGGGCTACCGGCTGTTCGAGAAGATTAGC 426 NLpep104 (w/o Met) A.A. VEGYRLFEKIS 427 NLpep105 (w/o Met) N.A. GTGGAGGGCTACCGGCTGTTCGAGCAGATTAGC 428 NLpep105 (w/o Met) A.A. VEGYRLFEQIS 429 NLpep106 (w/o Met) N.A. GTGGAGGGCTACCGGCTGTTCGAGAAGGAGAGC 430 NLpep106 (w/o Met) A.A. VEGYRLFEKES 431 NLpep107 (w/o Met) N.A. GTGGAGGGCTACCGGCTGTTCGAGCAGGAGAGC 432 NLpep107 (w/o Met) A.A. VEGYRLFEQES 433 NLpep108 (w/o Met) N.A. GTGGAGGGCTACCGGCTGTTCGAGCAGGAGCTG 434 NLpep108 (w/o Met) A.A. VEGYRLFEQEL 435 NLpep109 (w/o Met) N.A. ATTAGCGGCTGGCGGCTGATGAAGAACATTAGC 436 NLpep109 (w/o Met) A.A. ISGWRLMKNIS 437 NLpep110 (w/o Met) N.A. GTGGAGGGCTACCGGCTGTTCAAGAAGATTAGC 438 NLpep110 (w/o Met) A.A. VEGYRLFKKIS

In certain embodiments, a peptide from Table 1 is provided. In some embodiments, peptides comprise a single amino acid difference from GVTGWRLCKRISA (SEQ ID NO: 236) and/or any of the peptides listed in Table 1. In some embodiments, peptides comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid differences from GVTGWRLCKRISA (SEQ ID NO: 236) and/or any of the peptides listed in Table 1. In some embodiments, peptides are provided comprising one of the amino acid sequences of SEQ ID NOS: 3-438. In some embodiments, peptides are provided comprising one of the amino acid sequences of SEQ ID NOS: 3-438 with one or more additions, substitutions, and/or deletions. In some embodiments, a peptide or a portion thereof comprises greater than 70% sequence identity (e.g., 71%, 75%, 80%, 85%, 90%, 95%, 99%) with one or more of the amino acid sequence of SEQ ID NOS: 3-438. In some embodiments, nucleic acids are provided comprising one of the nucleic acid coding sequences of SEQ ID NOS: 3-438. In some embodiments, nucleic acids are provided comprising one of the nucleic acid sequences of SEQ ID NOS: 3-438 with one or more additions, substitutions, and/or deletions. In some embodiments, a nucleic acid or a portion thereof comprises greater than 70% sequence identity (e.g., 71%, 75%, 80%, 85%, 90%, 95%, 99%) with one or more of the nucleic acid sequence of SEQ ID NOS: 3-438. In some embodiments, nucleic acids are provided that code for one of the amino acid sequences of SEQ ID NOS: 3-438. In some embodiments, nucleic acids are provided that code for one of the amino acid sequences of SEQ ID NOS: 3-438 with one or more additions, substitutions, and/or deletions. In some embodiments, a nucleic acid is provided that codes for an amino acid with greater than 70% sequence identity (e.g., 71%, 75%, 80%, 85%, 90%, 95%, 99%) with one or more of the amino acid sequences of SEQ ID NOS: 3-438.

In certain embodiments, a nucleic acid from Table 1 is provided. In some embodiments, a nucleic acid encoding a peptide from Table 1 is provided. In some embodiments, a nucleic acid of the present invention codes for a peptide that comprises a single amino acid difference from MGVTGWRLCERILA (SEQ ID NO: 2) and/or any of the peptides listed in Table 1. In some embodiments, nucleic acids code for peptidespeptide comprising two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid differences from MGVTGWRLCERILA (SEQ ID NO: 2) and/or any of the peptides listed in Table 1. In some embodiments, nucleic acids are provided comprising the sequence of one of the nucleic acids in Table 1. In some embodiments, nucleic acids are provided comprising one of the nucleic acids of Table 1 with one or more additions, substitutions, and/or deletions. In some embodiments, a nucleic acid or a portion thereof comprises greater than 70% sequence identity (e.g., 71%, 75%, 80%, 85%, 90%, 95%, 99%) with one or more of the nucleic acids of Table 1.

In some embodiments, non-luminescent polypeptides that find use in embodiments of the present invention include polypeptides with one or more amino acid substitutions, deletions, or additions from SEQ ID NO: 440. In some embodiments, the present invention provides polypeptides comprising one or more of amino acid sequences of Table 2, and/or nucleic acids comprising the nucleic acid sequences of Table 2 (which code for the polypeptide sequences of Table 2).

TABLE 2 Polypeptide sequences SEQ ID NO Polymer ID 441 N.A. R11N 442 A.A R11N 443 N.A. T13I 444 A.A T13I 445 N.A. G15S 446 A.A G15S 447 N.A. L18Q 448 A.A L18Q 449 N.A. Q20K 450 A.A Q20K 451 N.A. V27M 452 A.A V27M 453 N.A. F31I 454 A.A F31I 455 N.A. F31L 456 A.A F31L 457 N.A. F31V 458 A.A F31V 459 N.A. Q32R 460 A.A Q32R 461 N.A. N33K 462 A.A N33K 463 N.A. N33R 464 A.A N33R 465 N.A. I56N 466 A.A I56N 467 N.A. V58A 468 A.A V58A 469 N.A. I59T 470 A.A I59T 471 N.A. G67S 472 A.A G67S 473 N.A. G67D 474 A.A G67D 475 N.A. K75E 476 A.A K75E 477 N.A. M106V 478 A.A M106V 479 N.A. M106I 480 A.A M106I 481 N.A. D108N 482 A.A D108N 483 N.A. R112Q 484 A.A R112Q 485 N.A. N144T 486 A.A N144T 487 N.A. L149M 488 A.A L149M 489 N.A. N156D 490 A.A N156D 491 N.A. N156S 492 A.A N156S 493 N.A. V157D 494 A.A V157D 495 N.A. V157S 496 A.A V157S 497 N.A. G8A 498 A.A G8A 499 N.A. G15A 500 A.A G15A 501 N.A. G25A 502 A.A G25A 503 N.A. G26A 504 A.A G26A 505 N.A. G35A 506 A.A G35A 507 N.A. G48A 508 A.A G48A 509 N.A. G51A 510 A.A G51A 511 N.A. G64A 512 A.A G64A 513 N.A. G67A 514 A.A G67A 515 N.A. G71A 516 A.A G71A 517 N.A. G95A 518 A.A G95A 519 N.A. G101A 520 A.A G101A 521 N.A. G111A 522 A.A G111A 523 N.A. G116A 524 A.A G116A 525 N.A. G122A 526 A.A G122A 527 N.A. G129A 528 A.A G129A 529 N.A. G134A 530 A.A G134A 531 N.A. G147A 532 A.A G147A 533 N.A. I54A 534 A.A I54A 535 N.A. 5A1 (G15A/D19A/G35A/G51A/G67A) 536 A.A 5A1 (G15A/D19A/G35A/G51A/G67A) 537 N.A. 4A1 (G15A/G35A/G67A/G71A) 538 A.A 4A1 (G15A/G35A/G67A/G71A) 539 N.A. 5A2 (G15A/G35A/G51A/G67A/G71A) 540 A.A 5A2 (G15A/G35A/G51A/G67A/G71A) 541 N.A. 5A2 + A15G 542 A.A 5A2 + A15G 543 N.A. 5A2 + A35G 544 A.A 5A2 + A35G 545 N.A. 5A2 + A51G 546 A.A 5A2 + A51G 547 N.A. 5A2 + A67G 548 A.A 5A2 + A67G 549 N.A. 5A2 + A71G 550 A.A 5A2 + A71G 551 N.A. 5A2 + R11A 552 A.A 5A2 + R11A 553 N.A. 5A2 + R11C 554 A.A 5A2 + R11C 555 N.A. 5A2 + R11D 556 A.A 5A2 + R11D 557 N.A. 5A2 + R11E 558 A.A 5A2 + R11E 559 N.A. 5A2 + R11F 560 A.A 5A2 + R11F 561 N.A. 5A2 + R11G 562 A.A 5A2 + R11G 563 N.A. 5A2 + R11H 564 A.A 5A2 + R11H 565 N.A. 5A2 + R11I 566 A.A 5A2 + R11I 567 N.A. 5A2 + R11K 568 A.A 5A2 + R11K 569 N.A. 5A2 + R11L 570 A.A 5A2 + R11L 571 N.A. 5A2 + R11M 572 A.A 5A2 + R11M 573 N.A. 5A2 + R11N 574 A.A 5A2 + R11N 575 N.A. 5A2 + R11P 576 A.A 5A2 + R11P 577 N.A. 5A2 + R11Q 578 A.A 5A2 + R11Q 579 N.A. 5A2 + R11S 580 A.A 5A2 + R11S 581 N.A. 5A2 + R11T 582 A.A 5A2 + R11T 583 N.A. 5A2 + R11V 584 A.A 5A2 + R11V 585 N.A. 5A2 + R11W 586 A.A 5A2 + R11W 587 N.A. 5A2 + R11Y 588 A.A 5A2 + R11Y 589 N.A. 5A2 + A15C 590 A.A 5A2 + A15C 591 N.A. 5A2 + A15D 592 A.A 5A2 + A15D 593 N.A. 5A2 + A15E 594 A.A 5A2 + A15E 595 N.A. 5A2 + A15F 596 A.A 5A2 + A15F 597 N.A. 5A2 + A15G 598 A.A 5A2 + A15G 599 N.A. 5A2 + A15H 600 A.A 5A2 + A15H 601 N.A. 5A2 + A15I 602 A.A 5A2 + A15I 603 N.A. 5A2 + A15K 604 A.A 5A2 + A15K 605 N.A. 5A2 + A15L 606 A.A 5A2 + A15L 607 N.A. 5A2 + A15M 608 A.A 5A2 + A15M 609 N.A. 5A2 + A15N 610 A.A 5A2 + A15N 611 N.A. 5A2 + A15P 612 A.A 5A2 + A15P 613 N.A. 5A2 + A15Q 614 A.A 5A2 + A15Q 615 N.A. 5A2 + A15R 616 A.A 5A2 + A15R 617 N.A. 5A2 + A15S 618 A.A 5A2 + A15S 619 N.A. 5A2 + A15T 620 A.A 5A2 + A15T 621 N.A. 5A2 + A15V 622 A.A 5A2 + A15V 623 N.A. 5A2 + A15W 624 A.A 5A2 + A15W 625 N.A. 5A2 + A15Y 626 A.A 5A2 + A15Y 627 N.A. 5A2 + L18A 628 A.A 5A2 + L18A 629 N.A. 5A2 + L18C 630 A.A 5A2 + L18C 631 N.A. 5A2 + L18D 632 A.A 5A2 + L18D 633 N.A. 5A2 + L18E 634 A.A 5A2 + L18E 635 N.A. 5A2 + L18F 636 A.A 5A2 + L18F 637 N.A. 5A2 + L18G 638 A.A 5A2 + L18G 639 N.A. 5A2 + L18H 640 A.A 5A2 + L18H 641 N.A. 5A2 + L18I 642 A.A 5A2 + L18I 643 N.A. 5A2 + L18K 644 A.A 5A2 + L18K 645 N.A. 5A2 + L18M 646 A.A 5A2 + L18M 647 N.A. 5A2 + L18N 648 A.A 5A2 + L18N 649 N.A. 5A2 + L18P 650 A.A 5A2 + L18P 651 N.A. 5A2 + L18Q 652 A.A 5A2 + L18Q 653 N.A. 5A2 + L18R 654 A.A 5A2 + L18R 655 N.A. 5A2 + L18S 656 A.A 5A2 + L18S 657 N.A. 5A2 + L18T 658 A.A 5A2 + L18T 659 N.A. 5A2 + L18V 660 A.A 5A2 + L18V 661 N.A. 5A2 + L18W 662 A.A 5A2 + L18W 663 N.A. 5A2 + L18Y 664 A.A 5A2 + L18Y 665 N.A. 5A2 + F31A 666 A.A 5A2 + F31A 667 N.A. 5A2 + F31C 668 A.A 5A2 + F31C 669 N.A. 5A2 + F31D 670 A.A 5A2 + F31D 671 N.A. 5A2 + F31E 672 A.A 5A2 + F31E 673 N.A. 5A2 + F31G 674 A.A 5A2 + F31G 675 N.A. 5A2 + F31H 676 A.A 5A2 + F31H 677 N.A. 5A2 + F31I 678 A.A 5A2 + F31I 679 N.A. 5A2 + F31K 680 A.A 5A2 + F31K 681 N.A. 5A2 + F31L 682 A.A 5A2 + F31L 683 N.A. 5A2 + F31M 684 A.A 5A2 + F31M 685 N.A. 5A2 + F31N 686 A.A 5A2 + F31N 687 N.A. 5A2 + F31P 688 A.A 5A2 + F31P 689 N.A. 5A2 + F31Q 690 A.A 5A2 + F31Q 691 N.A. 5A2 + F31R 692 A.A 5A2 + F31R 693 N.A. 5A2 + F31S 694 A.A 5A2 + F31S 695 N.A. 5A2 + F31T 696 A.A 5A2 + F31T 697 N.A. 5A2 + F31V 698 A.A 5A2 + F31V 699 N.A. 5A2 + F31W 700 A.A 5A2 + F31W 701 N.A. 5A2 + F31Y 702 A.A 5A2 + F31Y 703 N.A. 5A2 + V58A 704 A.A 5A2 + V58A 705 N.A. 5A2 + V58C 706 A.A 5A2 + V58C 707 N.A. 5A2 + V58D 708 A.A 5A2 + V58D 709 N.A. 5A2 + V58E 710 A.A 5A2 + V58E 711 N.A. 5A2 + V58F 712 A.A 5A2 + V58F 713 N.A. 5A2 + V58G 714 A.A 5A2 + V58G 715 N.A. 5A2 + V58H 716 A.A 5A2 + V58H 717 N.A. 5A2 + V58I 718 A.A 5A2 + V58I 719 N.A. 5A2 + V58K 720 A.A 5A2 + V58K 721 N.A. 5A2 + V58L 722 A.A 5A2 + V58L 723 N.A. 5A2 + V58M 724 A.A 5A2 + V58M 725 N.A. 5A2 + V58N 726 A.A 5A2 + V58N 727 N.A. 5A2 + V58P 728 A.A 5A2 + V58P 729 N.A. 5A2 + V58Q 730 A.A 5A2 + V58Q 731 N.A. 5A2 + V58R 732 A.A 5A2 + V58R 733 N.A. 5A2 + V58S 734 A.A 5A2 + V58S 735 N.A. 5A2 + V58T 736 A.A 5A2 + V58T 737 N.A. 5A2 + V58W 738 A.A 5A2 + V58W 739 N.A. 5A2 + V58Y 740 A.A 5A2 + V58Y 741 N.A. 5A2 + A67C 742 A.A 5A2 + A67C 743 N.A. 5A2 + A67D 744 A.A 5A2 + A67D 745 N.A. 5A2 + A67E 746 A.A 5A2 + A67E 747 N.A. 5A2 + A67F 748 A.A 5A2 + A67F 749 N.A. 5A2 + A67G 750 A.A 5A2 + A67G 751 N.A. 5A2 + A67H 752 A.A 5A2 + A67H 753 N.A. 5A2 + A67I 754 A.A 5A2 + A67I 755 N.A. 5A2 + A67K 756 A.A 5A2 + A67K 757 N.A. 5A2 + A67L 758 A.A 5A2 + A67L 759 N.A. 5A2 + A67M 760 A.A 5A2 + A67M 761 N.A. 5A2 + A67N 762 A.A 5A2 + A67N 763 N.A. 5A2 + A67P 764 A.A 5A2 + A67P 765 N.A. 5A2 + A67Q 766 A.A 5A2 + A67Q 767 N.A. 5A2 + A67R 768 A.A 5A2 + A67R 769 N.A. 5A2 + A67S 770 A.A 5A2 + A67S 771 N.A. 5A2 + A67T 772 A.A 5A2 + A67T 773 N.A. 5A2 + A67V 774 A.A 5A2 + A67V 775 N.A. 5A2 + A67W 776 A.A 5A2 + A67W 777 N.A. 5A2 + A67Y 778 A.A 5A2 + A67Y 779 N.A. 5A2 + M106A 780 A.A 5A2 + M106A 781 N.A. 5A2 + M106C 782 A.A 5A2 + M106C 783 N.A. 5A2 + M106D 784 A.A 5A2 + M106D 785 N.A. 5A2 + M106E 786 A.A 5A2 + M106E 787 N.A. 5A2 + M106F 788 A.A 5A2 + M106F 789 N.A. 5A2 + M106G 790 A.A 5A2 + M106G 791 N.A. 5A2 + M106H 792 A.A 5A2 + M106H 793 N.A. 5A2 + M106I 794 A.A 5A2 + M106I 795 N.A. 5A2 + M106K 796 A.A 5A2 + M106K 797 N.A. 5A2 + M106L 798 A.A 5A2 + M106L 799 N.A. 5A2 + M106N 800 A.A 5A2 + M106N 801 N.A. 5A2 + M106P 802 A.A 5A2 + M106P 803 N.A. 5A2 + M106Q 804 A.A 5A2 + M106Q 805 N.A. 5A2 + M106R 806 A.A 5A2 + M106R 807 N.A. 5A2 + M106S 808 A.A 5A2 + M106S 809 N.A. 5A2 + M106T 810 A.A 5A2 + M106T 811 N.A. 5A2 + M106V 812 A.A 5A2 + M106V 813 N.A. 5A2 + M106W 814 A.A 5A2 + M106W 815 N.A. 5A2 + M106Y 816 A.A 5A2 + M106Y 817 N.A. 5A2 + L149A 818 A.A 5A2 + L149A 819 N.A. 5A2 + L149C 820 A.A 5A2 + L149C 821 N.A. 5A2 + L149D 822 A.A 5A2 + L149D 823 N.A. 5A2 + L149E 824 A.A 5A2 + L149E 825 N.A. 5A2 + L149F 826 A.A 5A2 + L149F 827 N.A. 5A2 + L149G 828 A.A 5A2 + L149G 829 N.A. 5A2 + L149H 830 A.A 5A2 + L149H 831 N.A. 5A2 + L149I 832 A.A 5A2 + L149I 833 N.A. 5A2 + L149K 834 A.A 5A2 + L149K 835 N.A. 5A2 + L149M 836 A.A 5A2 + L149M 837 N.A. 5A2 + L149N 838 A.A 5A2 + L149N 839 N.A. 5A2 + L149P 840 A.A 5A2 + L149P 841 N.A. 5A2 + L149Q 842 A.A 5A2 + L149Q 843 N.A. 5A2 + L149R 844 A.A 5A2 + L149R 845 N.A. 5A2 + L149S 846 A.A 5A2 + L149S 847 N.A. 5A2 + L149T 848 A.A 5A2 + L149T 849 N.A. 5A2 + L149V 850 A.A 5A2 + L149V 851 N.A. 5A2 + L149W 852 A.A 5A2 + L149W 853 N.A. 5A2 + L149Y 854 A.A 5A2 + L149Y 855 N.A. 5A2 + V157A 856 A.A 5A2 + V157A 857 N.A. 5A2 + V157C 858 A.A 5A2 + V157C 859 N.A. 5A2 + V157D 860 A.A 5A2 + V157D 861 N.A. 5A2 + V157E 862 A.A 5A2 + V157E 863 N.A. 5A2 + V157F 864 A.A 5A2 + V157F 865 N.A. 5A2 + V157G 866 A.A 5A2 + V157G 867 N.A. 5A2 + V157H 868 A.A 5A2 + V157H 869 N.A. 5A2 + V157I 870 A.A 5A2 + V157I 871 N.A. 5A2 + V157K 872 A.A 5A2 + V157K 873 N.A. 5A2 + V157L 874 A.A 5A2 + V157L 875 N.A. 5A2 + V157M 876 A.A 5A2 + V157M 877 N.A. 5A2 + V157N 878 A.A 5A2 + V157N 879 N.A. 5A2 + V157P 880 A.A 5A2 + V157P 881 N.A. 5A2 + V157Q 882 A.A 5A2 + V157Q 883 N.A. 5A2 + V157R 884 A.A 5A2 + V157R 885 N.A. 5A2 + V157S 886 A.A 5A2 + V157S 887 N.A. 5A2 + V157T 888 A.A 5A2 + V157T 889 N.A. 5A2 + V157W 890 A.A 5A2 + V157W 891 N.A. 5A2 + V157Y 892 A.A 5A2 + V157Y 893 N.A. 5A2 + Q20K 894 A.A 5A2 + Q20K 895 N.A. 5A2 + V27M 896 A.A 5A2 + V27M 897 N.A. 5A2 + N33K 898 A.A 5A2 + N33K 899 N.A. 5A2 + V38I 900 A.A 5A2 + V38I 901 N.A. 5A2 + I56N 902 A.A 5A2 + I56N 903 N.A. 5A2 + D108N 904 A.A 5A2 + D108N 905 N.A. 5A2 + N144T 906 A.A 5A2 + N144T 907 N.A. 5A2 + V27M + A35G 908 A.A 5A2 + V27M + A35G 909 N.A. 5A2 + A71G + K75E 910 A.A 5A2 + A71G + K75E 911 N.A. 5A2 + R11E + L149M 912 A.A 5A2 + R11E + L149M 913 N.A. 5A2 + R11E + V157P 914 A.A 5A2 + R11E + V157P 915 N.A. 5A2 + D108N + N144T 916 A.A 5A2 + D108N + N144T 917 N.A. 5A2 + L149M + V157D 918 A.A 5A2 + L149M + V157D 919 N.A. 5A2 + L149M + V157P 920 A.A 5A2 + L149M + V157P 921 N.A. 3P (5A2 + R11E + L149M + V157P) 922 A.A 3P (5A2 + R11E + L149M + V157P) 923 N.A. 3P + D108N 924 A.A 3P + D108N 925 N.A. 3P + N144T 926 A.A 3P + N144T 927 N.A. 3E (5A2 + R11E + L149M + V157E) 928 A.A 3E (5A2 + R11E + L149M + V157E) 929 N.A. 3E + D108N 930 A.A 3E + D108N 931 N.A. 3E + N144T 932 A.A 3E + N144T 933 N.A. 5P (3P + D108N + N144T) 934 A.A 5P (3P + D108N + N144T) 935 N.A. 6P (5P + I56N) 936 A.A 6P (5P + I56N) 937 N.A. 5E (3E + D108N + N144T) 938 A.A 5E (3E + D108N + N144T) 939 N.A. 6E (5E + I56N) 940 A.A 6E (5E + I56N) 941 N.A. NLpoly1 (5A2 + R11N + A15S + L18Q + F31I + V58A + A67D + M106V + L149M + V157D) 942 A.A NLpoly1 (5A2 + R11N + A15S + L18Q + F31I + V58A + A67D + M106V + L149M + V157D) 943 N.A. NLpoly2 (5A2 + A15S + L18Q + F31I + V58A + A67D + M106V + L149M + V157D) 944 A.A NLpoly2 (5A2 + A15S + L18Q + F31I + V58A + A67D + M106V + L149M + V157D) 945 N.A. NLpoly3 (5A2 + R11N + L18Q + F31I + V58A + A67D + M106V + L149M + V157D) 946 A.A NLpoly3 (5A2 + R11N + L18Q + F31I + V58A + A67D + M106V + L149M + V157D) 947 N.A. NLpoly4 (5A2 + R11N + A15S + F31I + V58A + A67D + M106V + L149M + V157D) 948 A.A NLpoly4 (5A2 + R11N + A15S + F31I + V58A + A67D + M106V + L149M + V157D) 949 N.A. NLpoly5 (5A2 + R11N + A15S + L18Q + V58A + A67D + M106V + L149M + V157D) 950 A.A NLpoly5 (5A2 + R11N + A15S + L18Q + V58A + A67D + M106V + L149M + V157D) 951 N.A. NLpoly6 (5A2 + R11N + A15S + L18Q + F31I + A67D + M106V + L149M + V157D) 952 A.A NLpoly6 (5A2 + R11N + A15S + L18Q + F31I + A67D + M106V + L149M + V157D) 953 N.A. NLpoly7 (5A2 + R11N + A15S + L18Q + F31I + V58A + M106V + L149M + V157D) 954 A.A NLpoly7 (5A2 + R11N + A15S + L18Q + F31I + V58A + M106V + L149M + V157D) 955 N.A. NLpoly8 (5A2 + R11N + A15S + L18Q + F31I + V58A + A67D + L149M + V157D) 956 A.A NLpoly8 (5A2 + R11N + A15S + L18Q + F31I + V58A + A67D + L149M + V157D) 957 N.A. NLpoly9 (5A2 + R11N + A15S + L18Q + F31I + V58A + A67D + M106V + V157D) 958 A.A NLpoly9 (5A2 + R11N + A15S + L18Q + F31I + V58A + A67D + M106V + V157D) 959 N.A. NLpoly10 (5A2 + R11N + A15S + L18Q + F31I + V58A + A67D + M106V + L149M) 960 A.A NLpoly10 (5A2 + R11N + A15S + L18Q + F31I + V58A + A67D + M106V + L149M) 961 N.A. NLpoly11 (5A2 + A15S + L18Q + M106V + L149M + V157D) 962 A.A NLpoly11 (5A2 + A15S + L18Q + M106V + L149M + V157D) 963 N.A. NLpoly12 (5A2 + A15S + L18Q + A67D + M106V + L149M + V157D) 964 A.A NLpoly12 (5A2 + A15S + L18Q + A67D + M106V + L149M + V157D) 965 N.A. NLpoly13 (5A2 + R11N + A15S + L18Q + M106V + L149M + V157D) 966 A.A NLpoly13 (5A2 + R11N + A15S + L18Q + M106V + L149M + V157D) 967 N.A. 5P + V 968 A.A 5P + V 969 N.A. 5P + A 970 A.A 5P + A 971 N.A. 5P + VT 972 A.A 5P + VT 973 N.A. 5P + VA 974 A.A 5P + VA 975 N.A. 5P + AT 976 A.A 5P + AT 977 N.A. 5P + AA 978 A.A 5P + AA 979 N.A. 5P + GG 980 A.A 5P + GG 981 N.A. 5P + AA 982 A.A 5P + AA 983 N.A. 5P + ATG 984 A.A 5P + ATG 985 N.A. 5P + VTG 986 A.A 5P + VTG 987 N.A. 5P + VTA 988 A.A 5P + VTA 989 N.A. 5P + GTA 990 A.A 5P + GTA 991 N.A. 5P + VTGW 992 A.A 5P + VTGW 993 N.A. 5P + VTGWR 994 A.A 5P + VTGWR 995 N.A. 5P + VTGWE 996 A.A 5P + VTGWE 997 N.A. 5P + VTGWK 998 A.A 5P + VTGWK 999 N.A. 5P + VTGWQ 1000 A.A 5P + VTGWQ 1001 N.A. 5P + VTGWH 1002 A.A 5P + VTGWH 1003 N.A. 5P D1 (−157) 1004 A.A 5P D1 (−157) 1005 N.A. 5P D2 (−156-157) 1006 A.A 5P D2 (−156-157) 1007 N.A. 5P D3 (−155-157) 1008 A.A 5P D3 (−155-157) 1009 N.A. 5P D4 (−154-157) 1010 A.A 5P D4 (−154-157) 1011 N.A. 5P D5 (−153-157) 1012 A.A 5P D5 (−153-157) 1013 N.A. 5P D6 (−152-157) 1014 A.A 5P D6 (−152-157) 1015 N.A. 5P D7 (−151-157) 1016 A.A 5P D7 (−151-157) 1017 N.A. 5P + F31A 1018 A.A 5P + F31A 1019 N.A. 5P + F31C 1020 A.A 5P + F31C 1021 N.A. 5P + F31D 1022 A.A 5P + F31D 1023 N.A. 5P + F31E 1024 A.A 5P + F31E 1025 N.A. 5P + F31G 1026 A.A 5P + F31G 1027 N.A. 5P + F31H 1028 A.A 5P + F31H 1029 N.A. 5P + F31I 1030 A.A 5P + F31I 1031 N.A. 5P + F31K 1032 A.A 5P + F31K 1033 N.A. 5P + F31L 1034 A.A 5P + F31L 1035 N.A. 5P + F31M 1036 A.A 5P + F31M 1037 N.A. 5P + F31N 1038 A.A 5P + F31N 1039 N.A. 5P + F31P 1040 A.A 5P + F31P 1041 N.A. 5P + F31Q 1042 A.A 5P + F31Q 1043 N.A. 5P + F31R 1044 A.A 5P + F31R 1045 N.A. 5P + F31S 1046 A.A 5P + F31S 1047 N.A. 5P + F31T 1048 A.A 5P + F31T 1049 N.A. 5P + F31V 1050 A.A 5P + F31V 1051 N.A. 5P + F31W 1052 A.A 5P + F31W 1053 N.A. 5P + F31Y 1054 A.A 5P + F31Y 1055 N.A. 5P + L46A 1056 A.A 5P + L46A 1057 N.A. 5P + L46C 1058 A.A 5P + L46C 1059 N.A. 5P + L46D 1060 A.A 5P + L46D 1061 N.A. 5P + L46E 1062 A.A 5P + L46E 1063 N.A. 5P + L46F 1064 A.A 5P + L46F 1065 N.A. 5P + L46G 1066 A.A 5P + L46G 1067 N.A. 5P + L46H 1068 A.A 5P + L46H 1069 N.A. 5P + L46I 1070 A.A 5P + L46I 1071 N.A. 5P + L46K 1072 A.A 5P + L46K 1073 N.A. 5P + L46M 1074 A.A 5P + L46M 1075 N.A. 5P + L46N 1076 A.A 5P + L46N 1077 N.A. 5P + L46P 1078 A.A 5P + L46P 1079 N.A. 5P + L46Q 1080 A.A 5P + L46Q 1081 N.A. 5P + L46R 1082 A.A 5P + L46R 1083 N.A. 5P + L46S 1084 A.A 5P + L46S 1085 N.A. 5P + L46T 1086 A.A 5P + L46T 1087 N.A. 5P + L46V 1088 A.A 5P + L46V 1089 N.A. 5P + L46W 1090 A.A 5P + L46W 1091 N.A. 5P + L46Y 1092 A.A 5P + L46Y 1093 N.A. 5P + N108A 1094 A.A 5P + N108A 1095 N.A. 5P + N108C 1096 A.A 5P + N108C 1097 N.A. 5P + N108D 1098 A.A 5P + N108D 1099 N.A. 5P + N108E 1100 A.A 5P + N108E 1101 N.A. 5P + N108F 1102 A.A 5P + N108F 1103 N.A. 5P + N108G 1104 A.A 5P + N108G 1105 N.A. 5P + N108H 1106 A.A 5P + N108H 1107 N.A. 5P + N108I 1108 A.A 5P + N108I 1109 N.A. 5P + N108K 1110 A.A 5P + N108K 1111 N.A. 5P + N108L 1112 A.A 5P + N108L 1113 N.A. 5P + N108M 1114 A.A 5P + N108M 1115 N.A. 5P + N108P 1116 A.A 5P + N108P 1117 N.A. 5P + N108Q 1118 A.A 5P + N108Q 1119 N.A. 5P + N108R 1120 A.A 5P + N108R 1121 N.A. 5P + N108S 1122 A.A 5P + N108S 1123 N.A. 5P + N108T 1124 A.A 5P + N108T 1125 N.A. 5P + N108V 1126 A.A 5P + N108V 1127 N.A. 5P + N108W 1128 A.A 5P + N108W 1129 N.A. 5P + N108Y 1130 A.A 5P + N108Y 1131 N.A. 5P + T144A 1132 A.A 5P + T144A 1133 N.A. 5P + T144C 1134 A.A 5P + T144C 1135 N.A. 5P + T144D 1136 A.A 5P + T144D 1137 N.A. 5P + T144E 1138 A.A 5P + T144E 1139 N.A. 5P + T144F 1140 A.A 5P + T144F 1141 N.A. 5P + T144G 1142 A.A 5P + T144G 1143 N.A. 5P + T144H 1144 A.A 5P + T144H 1145 N.A. 5P + T144I 1146 A.A 5P + T144I 1147 N.A. 5P + T144K 1148 A.A 5P + T144K 1149 N.A. 5P + T144L 1150 A.A 5P + T144L 1151 N.A. 5P + T144M 1152 A.A 5P + T144M 1153 N.A. 5P + T144N 1154 A.A 5P + T144N 1155 N.A. 5P + T144P 1156 A.A 5P + T144P 1157 N.A. 5P + T144Q 1158 A.A 5P + T144Q 1159 N.A. 5P + T144R 1160 A.A 5P + T144R 1161 N.A. 5P + T144S 1440 A.A 5P + T144S 1163 N.A. 5P + T144V 1164 A.A 5P + T144V 1165 N.A. 5P + T144W 1166 A.A 5P + T144W 1167 N.A. 5P + T144Y 1168 A.A 5P + T144Y 1169 N.A. 5P + P157A 1170 A.A 5P + P157A 1171 N.A. 5P + P157C 1172 A.A 5P + P157C 1173 N.A. 5P + P157D 1174 A.A 5P + P157D 1175 N.A. 5P + P157E 1176 A.A 5P + P157E 1177 N.A. 5P + P157F 1178 A.A 5P + P157F 1179 N.A. 5P + P157G 1180 A.A 5P + P157G 1181 N.A. 5P + P157H 1182 A.A 5P + P157H 1183 N.A. 5P + P157I 1184 A.A 5P + P157I 1185 N.A. 5P + P157K 1186 A.A 5P + P157K 1187 N.A. 5P + P157L 1188 A.A 5P + P157L 1189 N.A. 5P + P157M 1190 A.A 5P + P157M 1191 N.A. 5P + P157N 1192 A.A 5P + P157N 1193 N.A. 5P + P157Q 1194 A.A 5P + P157Q 1195 N.A. 5P + P157R 1196 A.A 5P + P157R 1197 N.A. 5P + P157S 1198 A.A 5P + P157S 1199 N.A. 5P + P157T 1200 A.A 5P + P157T 1201 N.A. 5P + P157V 1202 A.A 5P + P157V 1203 N.A. 5P + P157W 1204 A.A 5P + P157W 1205 N.A. 5P + P157Y 1206 A.A 5P + P157Y 1207 N.A. 5P + I107L 1208 A.A 5P + I107L 1209 N.A. 5P + K75E 1210 A.A 5P + K75E 1211 N.A. 5P + K123E + N156D 1212 A.A 5P + K123E + N156D 1213 N.A. 5P + I76V 1214 A.A 5P + I76V 1215 N.A. 5P + G48D + H57R + L92M + I99V 1216 A.A 5P + G48D + H57R + L92M + I99V 1217 N.A. 5P + F31L + V36A + I99V 1218 A.A 5P + F31L + V36A + I99V 1219 N.A. 5P + F31L + H93P 1220 A.A 5P + F31L + H93P 1221 N.A. 5P + V90A 1222 A.A 5P + V90A 1223 N.A. 5P + I44V 1224 A.A 5P + I44V 1225 N.A. 5P + L46R + H86Q + M106V 1226 A.A 5P + L46R + H86Q + M106V 1227 N.A. 5P + R141H 1228 A.A 5P + R141H 1229 N.A. 5P + N33D + V58A 1230 A.A 5P + N33D + V58A 1231 N.A. 5P + I56N + P157H 1232 A.A 5P + I56N + P157H 1233 N.A. 5P + L46Q + P157H 1234 A.A 5P + L46Q + P157H 1235 N.A. 5P + I59V 1236 A.A 5P + I59V 1237 N.A. 5P + A51T + E74K + P113L 1238 A.A 5P + A51T + E74K + P113L 1239 N.A. 5P + V36A 1240 A.A 5P + V36A 1241 N.A. 5P + A51T 1242 A.A 5P + A51T 1243 N.A. 5P + H57R 1244 A.A 5P + H57R 1245 N.A. 5P + V58A 1246 A.A 5P + V58A 1247 N.A. 5P + E74K 1248 A.A 5P + E74K 1249 N.A. 5P + H86Q 1250 A.A 5P + H86Q 1251 N.A. 5P + H93P 1252 A.A 5P + H93P 1253 N.A. 5P + I99V 1254 A.A 5P + I99V 1255 N.A. 5P + K123E 1256 A.A 5P + K123E 1257 N.A. 5P + T128S 1258 A.A 5P + T128S 1259 N.A. 5P + L142Q + T154N 1260 A.A 5P + L142Q + T154N 1261 N.A. 5P + H57Q 1262 A.A 5P + H57Q 1263 N.A. 5P + L92M 1264 A.A 5P + L92M 1265 N.A. 5P + P113L 1266 A.A 5P + P113L 1267 N.A. 5P + G48D 1268 A.A 5P + G48D 1269 N.A. 5P − B9 (−147-157) 1270 A.A 5P − B9 (−147-157) 1271 N.A. 5P + L46R + P157S 1272 A.A 5P + L46R + P157S 1273 N.A. 5P + L46H + P157H 1274 A.A 5P + L46H + P157H 1275 N.A. 5P + L46R + H93P 1276 A.A 5P + L46R + H93P 1277 N.A. 5P + L46R + H93P + F31L 1278 A.A 5P + L46R + H93P + F31L 1279 N.A. 5P + L46R + H93P + K75E 1280 A.A 5P + L46R + H93P + K75E 1281 N.A. 5P + L46R + H93P + I76V 1282 A.A 5P + L46R + H93P + I76V 1283 N.A. 8S (5P + L46R + H93P + P157S + F31L) 1284 A.A 8S (5P + L46R + H93P + P157S + F31L) 1285 N.A. 5P + L46R + H93P + P157S + K75E 1286 A.A 5P + L46R + H93P + P157S + K75E 1287 N.A. 5P + L46R + H93P + P157S + I76V 1288 A.A 5P + L46R + H93P + P157S + I76V 1289 N.A. 12S (8S + A51T + K75E + I76V + I107L) 1290 A.A 12S (8S + A51T + K75E + I76V + I107L) 1291 N.A. 11S (12-A51T) 1292 A.A 11S (12-A51T) 1293 N.A. 12S-K75E 1294 A.A 12S-K75E 1295 N.A. 12S-I76V 1296 A.A 12S-I76V 1297 N.A. 12S-I107L 1298 A.A 12S-I107L

The polypeptides and coding nucleic acid sequences of Table 2 (SEQ ID NOS: 441-1298) all contain N-terminal Met residues (amino acids) or ATG start codons (nucleic acids). In some embodiments, the polypeptides and coding nucleic acid sequences of Table 2 are provided without N-terminal Met residues or ATG start codons (SEQ ID NOS: 1299-2156).

In certain embodiments, a polypeptide of one of the amino acid polymers of SEQ ID NOS: 441-2156 is provided. In some embodiments, polypeptides comprise a single amino acid difference from SEQ ID NO: 440. In some embodiments, polypeptides comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 . . . 35 . . . 40 . . . 45 . . . 50, or more) amino acid differences from SEQ ID NO: 440 and/or any of the amino acid polymers of SEQ ID NOS:441-2156. In some embodiments, polypeptides are provided comprising the sequence of one of the amino acid polymers of SEQ ID NOS: 441-2156 with one or more additions, substitutions, and/or deletions. In some embodiments, a polypeptide or a portion thereof comprises greater than 70% sequence identity (e.g., >71%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%) with one or more of the amino acid polymers of SEQ ID NOS: 441-2156.

In certain embodiments, a nucleic acid from Table 2 is provided. In some embodiments, a nucleic acid encoding a polypeptide from Table 2 is provided. In some embodiments, a nucleic acid of the present invention codes for a polypeptide that comprises a single amino acid difference from SEQ ID NO: 440 and/or any of the amino acid polymers of SEQ ID NOS: 441-2156. In some embodiments, nucleic acids code for a polypeptide comprising two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 . . . 35 . . . 40 . . . 45 . . . 50, or more) amino acid differences from SEQ ID NO: 440 and/or any of the polypeptides listed in Table 2. In some embodiments, nucleic acids are provided comprising the sequence of one of the nucleic acid polymers of SEQ ID NOS: 441-2156. In some embodiments, nucleic acids are provided comprising the sequence of one of the nucleic acid polymers of SEQ ID NOS: 441-2156 with one or more additions, substitutions, and/or deletions. In some embodiments, a nucleic acid or a portion thereof comprises greater than 70% sequence identity (e.g., >71%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%) with one or more of the nucleic acid polymers of SEQ ID NOS: 441-2156. In some embodiments, a nucleic acid or a portion thereof codes for an polypeptide comprising greater than 70% sequence identity (e.g., >71%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%) with one or more of the amino acid polymers of SEQ ID NOS: 441-2156. In some embodiments, nucleic acids are provided that code for one of the polypeptides of SEQ ID NOS: 441-2156. In some embodiments, nucleic acids are provided that code for one of the polypeptides of SEQ ID NOS: 441-2156 with one or more additions, substitutions, and/or deletions.

In some embodiments, a non-luminescent peptide or polypeptide and/or an interaction element, comprises a synthetic peptide, peptide containing one or more non-natural amino acids, peptide mimetic, conjugated synthetic peptide (e.g., conjugated to a functional group (e.g., fluorophore, luminescent substrate, etc.)).

The present invention provides compositions and methods that are useful in a variety of fields including basic research, medical research, molecular diagnostics, etc. Although the reagents and assays described herein are not limited to any particular applications, and any useful application should be viewed as being within the scope of the present invention, the following are exemplary assays, kits, fields, experimental set-ups, etc. that make use of the presently claimed invention.

Typical applications that make use of embodiments of the present invention involve the monitoring/detection of protein dimerization (e.g., heterodimers, homodimers), protein-protein interactions, protein-RNA interactions, protein-DNA interactions, nucleic acid hybridization, protein-small molecule interactions, or any other combinations of molecular entities. A first entity of interest is attached to a first member of a non-luminescent pair and the second entity of interest is attached to the second member of a non-luminescent pair. If a detectable signal is produced under the particular assay conditions, then interaction of the first and second entities are inferred. Such assays are useful for monitoring molecular interactions under any suitable conditions (e.g., in vitro, in vivo, in situ, whole animal, etc.), and find use in, for example, drug discovery, elucidating molecular pathways, studying equilibrium or kinetic aspects of complex assembly, high throughput screening, proximity sensor, etc.

In some embodiments, a non-luminescent pair of known characteristics (e.g., spectral characteristics, mutual affinity of pair) is used to elucidate the affinity of, or understand the interaction of, an interaction pair of interest. In other embodiments, a well-characterized interaction pair is used to determine the characteristics (e.g., spectral characteristics, mutual affinity of pair) of a non-luminescent pair.

Embodiments described herein may find use in drug screening and/or drug development. For example, the interaction of a small molecule drug or an entire library of small molecules with a target protein of interest (e.g., therapeutic target) is monitored under one or more relevant conditions (e.g., physiological conditions, disease conditions, etc.). In other embodiments, the ability of a small molecule drug or an entire library of small molecules to enhance or inhibit the interactions between two entities (e.g., receptor and ligand, protein-protein, etc.) is assayed. In some embodiments, drug screening applications are carried out in a high through-put format to allow for the detection of the binding of tens of thousands of different molecules to a target, or to test the effect of those molecules on the binding of other entities.

In some embodiments, the present invention provides the detection of molecular interactions in living organisms (e.g., bacteria, yeast, eukaryotes, mammals, primates, human, etc.) and/or cells. In some embodiments, fusion proteins comprising signal and interaction (target) polypeptides are co-expressed in the cell or whole organism, and signal is detected and correlated to the formation of the interaction complex. In some embodiments, cells are transiently and/or stably transformed or transfected with vector(s) coding for non-luminescent element(s), interaction element(s), fusion proteins (e.g., comprising a signal and interaction element), etc. In some embodiments, transgenic organisms are generated that code for the necessary fusion proteins for carrying out the assays described herein. In other embodiments, vectors are injected into whole organisms. In some embodiments, a transgenic animal or cell (e.g., expressing a fusion protein) is used to monitor the biodistribution of a small molecule or a biologic tethered (e.g., conjugated or genetically fused) to NLpeptide sequence that would form a complex in the subcellular compartments and/or tissues where it concentrates.

The compositions and methods provided herein, as well as any techniques or technologies based thereon find use in a variety of applications and fields, a non-limiting list of example applications follows:

-   -   Antibody-free Western Blot: For example, a protein of interest         is fused to a non-luminescent peptide (e.g., by genetic         engineering) and expressed by any suitable means. The proteins         separated (e.g., by PAGE) and transferred to a membrane. The         membrane is then washed with complimentary non-luminescent         polypeptide (e.g. allowing a luminescent complex to form), and         placed on imager (e.g., utilizing a CCD camera) with Furimazine         (PBI-3939) atop the membrane, and the protein of interest is         detected (e.g., via the luminescence of the luminescent         complex).     -   “LucCytochemistry”: For example, a protein of interest is         expressed fused to a non-luminescent peptide or polypeptide and         then detected with a complimentary non-luminescent polypeptide         or peptide in a fashion analogous to immunocytochemistry.     -   Protein localization assay: For example, a localization signal         is added to a non-luminescent polypeptide or polypeptide (e.g.,         via genetic engineering) and expressed in cells (e.g., a nuclear         localization signal added would result in expression of the         non-luminescent polypeptide in the nucleus). A complimentary         non-luminescent peptide or polypeptide is fused to a protein of         interest (e.g., via genetic engineering) and expressed in cells         with the non-luminescent polypeptide or peptide. Luminescence is         produced if the protein of interest localizes in the same         subcellular compartment (e.g., the nucleus) as the         signal-localized non-luminescent polypeptide.     -   Protein Stability Assay: For example, a protein of interest is         fused to a non-luminescent peptide or polypeptide (e.g., via         genetic engineering) and incubated under one or more conditions         of interest. A complimentary non-luminescent polypeptide or         peptide is added (e.g., at various time points), and         luminescence is used to quantify the amount of protein of         interest (e.g., a proxy for stability).     -   Protein Detection/Quantification: For example, a protein of         interest fused to a non-luminescent peptide or polypeptide         (e.g., via genetic engineering) and expressed and/or manipulated         by any method. The complimentary non-luminescent polypeptide or         peptide is then added to detect and/or quantify the protein of         interest.     -   Protein Purification: For example, a protein of interest is         fused to a non-luminescent peptide or polypeptide (e.g., via         genetic engineering) and expressed by any method. The mixture of         proteins is passed through an immobilized complimentary         non-luminescent polypeptide or peptide (e.g., on beads, on a         column, on a chip, etc.), washed with suitable buffer and eluted         (e.g., with a buffer of high ionic strength or low pH). A mutant         form of the non-luminescent peptide or polypeptide that does not         activate the luminescence of the complimentary non-luminescent         peptide or polypeptide may be used to elute the protein of         interest.     -   Pull-down: For example, an immobilized, complimentary,         non-luminescent polypeptide is used to isolate a protein of         interest (and interacting proteins) that is fused to a         non-luminescent peptide (e.g., via genetic engineering).     -   G-Coupled Protein Receptor (GPCR) Internalization Assay: For         example, a non-luminescent peptide or polypeptide is fused to a         GPCR of interest (e.g., via genetic engineering) and expressed         on the surface of cells. A complimentary non-luminescent         polypeptide or peptide is added to the media of the cells and         used to detect the GPCR on cell surface. A ligand is added to         stimulate the internalization of the GPCR, and a decrease in         luminescence is observed.     -   Membrane Integrity Assay for Cell Viability: For example, when         the cell membrane of a cell expressing a non-luminescent         polypeptide become compromised, a non-luminescent peptide enters         the cell (e.g., a peptide that otherwise can't cross the cell         membrane), thereby forming a luminescent complex, and generating         luminescence.     -   5-Hydroxymethyl Cytosine Detection: For example, a cysteine is         added to a non-luminescent peptide and incubated with DNA and a         methyltransferase. The methyltransferase catalyzes the addition         of the thiol (cysteine) only onto cytosine residues that are         5-hydroxymethylated. Unincorporated peptide is then separated         from the DNA (using any method possible), and a non-luminescent         polypeptide is added to detect the peptide conjugated to the         DNA.     -   Formyl Cytosine Detection: For example, similar to the         5-hydroxymethyl cytosine detection above, this detection method         uses chemistry with specific reactivity for formyl cytosine.     -   Viral Incorporation: Nucleic acid coding for a non-luminescent         peptide or polypeptide is incorporated into a viral genome, and         the complementary non-luminescent polypeptide or peptide is         constitutively expressed in the target cells. Upon infection of         the target cells and expression of the non-luminescent peptide,         the bioluminescent complex forms and a signal is detected (e.g.,         in the presence of substrate).     -   Chemical Labeling of Proteins: A non-luminescent peptide is         fused or tethered to a reactive group (e.g., biotin,         succinimidyl ester, maleimide, etc.). A protein of interest         (e.g., antibody) is tagged with the non-luminescent peptide         through binding of the reactive group to the protein of         interest. Because the peptide is small, it does not affect the         functionality of the protein of interest. Complimentary         non-luminescent polypeptide is added to the system, and a         luminescent complex is produced upon binding to the polypeptide         to the peptide.

The above applications of the compositions and methods of the present invention are not limiting and may be modified in any suitable manner while still being within the scope of the present invention.

The present invention also provides methods for the design and/or optimization of non-luminescent pairs/groups and the bioluminescent complexes that form therefrom. Any suitable method for the design of non-luminescent pairs/groups that are consistent with embodiments described herein, and/or panels thereof, is within the scope of the present invention.

In certain embodiments, non-luminescent pairs/groups are designed de novo to lack luminescence individually and exhibit luminescence upon association. In such embodiments, the strength of the interaction between the non-luminescent elements is insufficient to produce a bioluminescent signal in the absence of interaction elements to facilitate formation of the bioluminescent complex.

In other embodiments, non-luminescent elements and/or non-luminescent pairs are rationally designed, for example, using a bioluminescent protein (e.g., SEQ ID NO: 2157) as a starting point. For example, such methods may comprise: (a) aligning the sequences of three or more related proteins; (b) determining a consensus sequence for the related proteins; (c) providing first and second fragments of a bioluminescent protein that is related to the ones from which the consensus sequence was determined, wherein the fragments are individually substantially non-luminescent but exhibit luminescence upon interaction of the fragments; (d) mutating the first and second fragments at one or more positions each (e.g., in vitro, in silico, etc.), wherein said mutations alter the sequences of the fragments to be more similar to a corresponding portion of the consensus sequence, wherein the mutating results in a non-luminescent pair that are not fragments of a preexisting protein, and (e) testing the non-luminescent pair for the absence of luminescence when unassociated and luminescence upon association of the non-luminescent pair. In other embodiments, first and second fragments of one of the proteins used in determining the consensus sequence are provided, mutated, and tested.

In some embodiments, the above methods are not limited to the design and/or optimization of non-luminescent pairs. The same steps are performed to produce pairs of elements that lack a given functionality (e.g., enzymatic activity) individually, but display such functionality when associated. In any of these cases, the strength of the interaction between the non-luminescent pair elements may be altered via mutations to ensure that it is insufficient to produce functionality in the absence of interaction elements that facilitate formation of the bioluminescent complex.

EXPERIMENTAL Example 1 Generation of Peptides

Peptide constructs were generated by one of three methods: annealing 5′-phosphorylated oligonucleotides followed by ligation to pF4Ag-Barnase-HALOTAG vector (Promega Corporation; cut with SgfI and XhoI) or pFN18A (Promega Corporation; cut with SgfI and XbaI), site directed mutagenesis using Quik Change Lightning Multi kit from Agilent or outsourcing the cloning to Gene Dynamics.

Example 2 Peptide Preparation

The peptides generated in Example 1 were prepared for analysis by inoculating a single colony of KRX E. coli cells (Promega Corporation) transformed with a plasmid encoding a peptide into 2-5 ml of LB culture and grown at 37° C. overnight. The overnight cultures (10 ml) were then diluted into 1 L of LB and grown at 37° C. for 3 hours. The cultures were then induced by adding 10 ml 20% rhamnose to the 1 L culture and induced at 25° C. for 18 hours.

After induction, 800 ml of each culture was spun at 5000×g at 4° C. for 30 minutes. The pellet generated was then resuspended in 80 ml Peptide Lysis Buffer (25 mM HEPES pH 7.4, 0.1× Passive Lysis Buffer (Promega Corporation), 1 ml/ml lysozyme and 0.03 U/μl RQ1 DNase (Promega Corporation)) and incubated at room temperature for 15 minutes. The lysed cells were then frozen on dry ice for 15 minutes and then thawed in a room temperature bath for 15 minutes. The cells were then spun at 3500×g at 4° C. for 30 minutes. The supernatants were aliquoted into 10 ml samples with one aliquot of 50 μl placed into a 1.5 ml tube.

To the 50 μl samples, 450 μl H₂O and 167 μl 4×SDS Loading Dye were added, and the samples incubated at 95° C. for 5 minutes. After heating, 5 μl of each sample was loaded (in triplicate) onto an SDS-PAGE gel, and the gel run and stained according to the manufacturer's protocol. The gel was then scanned on a Typhoon Scanner (excitation 532 nm, emission 580 nm, PMT sensitivity 400V). The resulting bands were quantified using the ImageQuant (5.2) software. Each of the three replicate intensities was averaged, and the average intensity of NLpep53-HT was defined at 12× concentration. The concentrations of all other peptides were relative to Pep53-HT.

Example 3 Peptide Analysis

All of the peptides generated in Examples 1-2 contained single mutations to the peptide sequence: GVTGWRLCKRISA (SEQ ID NO: 236). All of the peptides were fused to a HALOTAG protein (Promega Corporation). Peptides identified as “HT-NLpep” indicate that the peptide is located at the C-terminus of the HALOTAG protein. In this case, the gene encoding the peptide includes a stop codon, but does not include a methionine to initiate translation. Peptides identified as “NLpep-HT” indicate that the peptide is at the N-terminus of the HALOTAG protein. In this case, the peptide does include a methionine to initiate translation, but does not include a stop codon.

To determine the ability of the peptides to activate luminescence, individual colonies of KRX E. coli cells (Promega Corporation) was transformed with a plasmid encoding a peptide from Example 1, inoculated in 200 μl of minimal medium (1× M9 salts, 0.1 mM CaCl₂, 2 mM MgSO₄, 1 mM Thiamine HCl, 1% gelatin, 0.2% glycerol, and 100 ul/ml Ampicillin) and grown at 37° C. overnight. In addition to the peptides, a culture of KRX E. coli cells expressing a wild-type (WT) fragment of residues 1-156 of the NanoLuc was grown. All peptides and the WT fragment were inoculated into at least 3 separate cultures.

After the first overnight growth, 10 μl of culture was diluted into 190 μl fresh minimal medium and again grown at 37° C. overnight.

After the second overnight growth, 10 μl of the culture was diluted into 190 μl of auto-induction medium (minimal medium+5% glucose and 2% rhamnose). The cultures were then inducted at 25° C. for approximately 18 hours.

After induction, the small peptide mutant cultures were assayed for activity. The cultures containing the WT 1-156 fragment were pooled, mixed with 10 ml of 2× Lysis Buffer (50 mM HEPES pH 7.4, 0.3× Passive Lysis Buffer, and 1 mg/ml lysozyme) and incubated at room temperature for 10 minutes. 30 μl of the lysed WT 1-156 culture was then aliquoted into wells of a white, round bottom 96-well assay plate (Costar 3355). To wells of the assay plate, 20 μl of a peptide culture was added, and the plate incubated at room temperature for 10 minutes. After incubation, 50 μl NANOGLO Luciferase Assay Reagent (Promega Corporation) was added, and the samples incubated at room temperature for 10 minutes. Luminescence was measured on a GLOMAX luminometer with 0.5 s integrations.

The results (See Table 3 and FIG. 1) demonstrate various mutations in the peptide (relative to SEQ ID NO: 1) that altered (e.g., increased, decreased) the luminescence following complementation with the wild-type non-luminescent polypeptide. The increased luminescence is thought to stem from one (or a combination) of five main factors, any of which are beneficial: affinity between the non-luminescent peptide and non-luminescent polypeptide, expression of the peptide, intracellular solubility, intracellular stability, and bioluminescent activity. The present invention though is not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice the present invention.

TABLE 3 Mutation HT-NLPep NLpep-HT HT-Pep st. dev. Pep-HT st.dev. G157D 0.1137 0.5493 N.D. N.D. G157N 0.6415 3.3074 0.2512 1.4828 G157S 1.9937 1.7156 0.8554 1.0563 G157E 0.1959 1.4461 0.0811 0.3221 G157H 0.9380 0.5733 0.4366 0.2277 G157C N.D. 0.0468 N.D. 0.0081 G157P N.D. 0.0543 N.D. 0.0106 V158I 0.6075 1.6010 0.3283 0.6264 V158A 0.1348 0.1438 0.0561 0.0447 V158K 0.0770 0.1923 0.0323 0.0521 V158Q 0.0445 0.0397 0.0188 0.0160 V158S 0.0487 0.0838 0.0189 0.0251 T159V 0.5658 0.0455 0.2293 0.0005 T159K 0.0490 0.0307 0.0120 0.0103 T159Q 0.3979 0.0310 0.1063 0.0091 W161T 0.0028 0.0100 0.0007 0.0049 W161K 0.0002 0.0008 9.7E−06 0.0001 W161V 0.0086 0.0050 0.0062 0.0016 W161F N.D. 0.0717 N.D. 0.0049 W161Y N.D. 0.2154 N.D. 0.0103 W161E N.D. 0.0012 N.D. 0.0002 L163I N.D. 0.2923 N.D. 0.1198 L163V 0.1727 0.1190 0.0257 0.0288 L163T 0.0259 0.0262 0.0077 0.0122 L163Y 0.0512 0.1959 0.0126 0.1043 L163K 0.0885 0.0786 0.0130 0.0244 C164N 0.0874 0.1081 0.0097 0.0160 C164T 0.0116 0.0084 0.0029 0.0013 C164F N.D. 13.3131 N.D. 3.6429 C164Y N.D. 1.0092 N.D. 0.2592 C164S N.D. 0.0202 N.D. 0.0029 C164H N.D. 0.7597 N.D. 0.2149 C164M N.D. 3.2618 N.D. 1.1763 C164A N.D. 0.0858 N.D. 0.0196 C164Q N.D. 0.0211 N.D. 0.0044 C164L N.D. 1.0170 N.D. 0.2464 C164K N.D. 0.0005 N.D. 0.0001 R166K 1.0910 1.2069 0.2266 0.5913 R166N 0.1033 0.1182 0.0289 0.0542 I167V 0.8770 1.0824 0.1113 0.2642 I167Q 0.0178 0.1172 0.0252 0.0150 I167E 0.2771 0.2445 0.0358 0.0456 I167R 0.0464 0.0469 0.0027 0.0084 I167F 0.2832 0.1793 0.0159 0.0683 A169N 0.9115 1.7775 0.1114 0.5901 A169T 0.9448 1.3720 0.0930 0.6021 A169R 0.9851 0.5014 0.2205 0.1895 A169L 1.1127 0.9047 0.1906 0.2481 A169E 0.8457 0.7889 0.1445 0.0819

Example 4 Generation of Non-Luminescent Polypeptides

Using pF4Ag-NanoLuc1-156 (WT 1-156) as a template, error-prone PCR (epPCR) was performed using the Diversify PCR Random Mutagenesis Kit from Clontech. The resulting PCR product was digested with SgfI and XbaI and ligated to pF4Ag-Barnase (Promega Corporation), a version of the commercially-available pF4A vector (Promega) which contains T7 and CMV promoters and was modified to contain an E. coli ribosome-binding site. Following transformation into KRX E. coli cells (Promega Corporation) by heat shock at 42° C., individual colonies were used to inoculate 200 μl cultures in clear, flat bottom 96-well plates (Costar 3370).

Example 5 Non-Luminescent Polypeptide Analysis

To determine the luminescence of the non-luminescent polypeptide mutants generated in Example 4, individual colonies of the KRX E. coli cells (Promega Corporation) transformed with a plasmid containing one of the non-luminescent polypeptide mutants from Example 4 was grown according to the procedure used in Example 3. The bacterial cultures were also induced according to the procedure used in Example 3.

To assay each non-luminescent polypeptide mutant induced culture, 30 μl of assay lysis buffer (25 mM HEPES pH 7.4, 0.3× Passive Lysis Buffer (Promega Corporation)), 0.006 U/μl RQ1 DNase (Promega Corporation) and 1× Peptide Solution (the relative concentration of the peptides were determined as explained in Example 2; from the relative concentration determined, the peptides were diluted to 1× in the lysis buffer) containing either the peptide fragment GVTGWRLCKRISA (SEQ ID NO: 18) or GVTGWRLFKRISA (SEQ ID NO: 106) were aliquoted into wells of a 96-well assay plate (Costar 3355). To the wells of the assay plate, 20 μl of an induced non-luminescent polypeptide mutant culture was added, and the plate incubated at room temperature for 10 minutes. After incubation, 50 μl of NANOGLO Luciferase Assay Reagent (Promega Corporation) was added, and the samples incubated at room temperature for 10 minutes. Luminescence was measured on a GLOMAX luminometer with 0.5 s integrations.

The results (Table 4 and FIG. 2) demonstrate numerous point mutations that improve the luminescence of the non-luminescent polypeptide upon complementation with two different peptides. Similar to the mutations in the peptide, these mutations in the non-luminescent polypeptide may stem from various factors, all of which are beneficial to the system as a whole.

TABLE 4 Mutation V157D F31I L18Q R11N GVTGWRLCKRISA 4.98 4.1 3.81 3.37 st dev 0.48 0.37 0.29 0.67 GVTGWRLFKRISA 3.02 2.83 2.99 2.09 st dev 0.77 0.61 0.82 0.03 Mutation Q32R M106V M106I G67S GVTGWRLCKRISA 1.52 1.3 1.27 1.22 st dev 0.2 0.22 0.04 0.26 GVTGWRLFKRISA 1.04 1.4 1.31 1.29 st dev 0.19 0.25 0.35 0.22 Mutation F31L L149M N33K I59T GVTGWRLCKRISA 3.13 2.89 2.15 1.07 st dev 0.26 0.39 0.2 0.07 GVTGWRLFKRISA 2.86 2.16 1.76 1.35 st dev 0.7 0.26 0.08 0.37 Mutation I56N T13I F31V N33R GVTGWRLCKRISA 0.44 2.18 2.12 2.1 st dev 0.05 0.75 0.09 0.18 GVTGWRLFKRISA 1.81 1.44 2.12 1.56 st dev 0.35 0.34 0.46 0.16 Mutation V27M Q20K V58A K75E GVTGWRLCKRISA 1.99 4.43 1.88 2.08 st dev 0.09 0.84 0.6 0.47 GVTGWRLFKRISA 1.7 2.33 1.07 2.05 st dev 0.11 0.38 0.26 0.37 Mutation G15S G67D R112N N156D GVTGWRLCKRISA 1.98 1.78 1.61 1.57 st dev 0.99 0.11 0.2 0.21 GVTGWRLFKRISA 2.34 1.57 1.45 1.21 st dev 0.82 0.17 0.47 0.26 Mutation D108N N144T N156S GVTGWRLCKRISA 2.08 3.69 1.04 st dev 0.6 1.12 0.29 GVTGWRLFKRISA 1.88 2.26 1.4 st dev 0.38 0.51 0.28 *Units in Table 4 are RLU(mutant)/RLU(WT)

Example 6 Glycine to Alanine Substitutions in Non-Luminescent Polypeptide

The following example identified glycine residues within the non-luminescent polypeptide that can be substituted to alanine to provide an improved (e.g., greater luminescent signal) non-luminescent polypeptide. The substitutions were made singly (See FIG. 3), or in composites (FIG. 2). Non-luminescent polypeptides containing glycine to alanine substitutions were generated as described in Example 1.

Each single mutant colony was inoculated in 200 μl Minimal Media (lx M9 salts, 0.1 mM CaCl₂, 2 mM MgSO₄, 1 mM Thiamine HCl, 1% gelatin, 0.2% glycerol and 1× ampicillin) and incubated with shaking at 37° C. for 20 hours. 10 μl of the culture was then added to 190 μl of fresh Minimal Media and incubated again with shaking at 37° C. for 20 hours. 10 μl of the second culture was then added to 190 μl Auto-Induction Media (Minimal Media+5% glucose+2% rhamnose) and incubated with shaking at 25° C. for 18 hours to allow expression of the non-luminescent polypeptide.

To assay each mutant culture, 30 μl of assay lysis buffer (50 mM HEPES pH 7.5, 0.3× Passive Lysis Buffer (Promega Corporation)) and 0.006 U/μl RQ1 DNase (Promega Corporation)) containing non-luminescent peptide (1:10 dilution of NLpep9-HT (NLpep9 is SEQ ID NO: 17 and 18; HT is HaloTag E. coli clarified lysate) was added. The samples were shaken at room temperature for 10 minutes, and then 50 μl NANOGLO Luciferase Assay Reagent (Promega Corporation) was added. The samples were incubated at room temperature for 10 minutes, and luminescence was measured on a GLOMAX luminometer with 0.5 s integrations.

To generate the NLpep9-HT E. coli clarified lysate, 5 ml LB was inoculated with a single E. coli colony of NLpep9-HT and incubated at 37° C. overnight. 500 μl of the overnight culture was then diluted in 50 mls LB and incubated at 37° C. for 3 hours. 500 μl of 20% rhamnose was added and incubated at 25° C. for 18 hours. The expression culture was centrifuged at 3000×g for 30 minutes, and the cell pellet resuspended in 5 ml peptide lysis buffer (25 mM HEPES, pH 7.5, 0.1× Passive Lysis Buffer, 1 mg/ml lysozyme, and 0.3 U/μl RQ1 DNase) and incubated at room temperature for 10 minutes. The lysed sample was placed on dry ice for 15 minutes, thawed in a room temperature water bath and centrifuged at 3500×g for 30 minutes. The supernatant was the clarified lysate.

FIGS. 3 and 4 demonstrate the effects of the mutations on luminescence.

Example 7 Mutations in Non-Luminescent Peptide

In the following example, mutations were made in the non-luminescent peptide based on alignment to other fatty acid binding proteins (FABPs) and were chosen based on high probability (frequency in FABPs) to identify a mutation that retains/improves activity (such as NLpep2, 4, and 5) or establish that a mutation is not likely to be tolerated at that position (such as NLpep3). NLpep1-5 contain single mutations (See Table 1), and NLpep6-9 are composite sets of the mutations in NLpep2, 4, and 5 (See Table 1). Mutants were generated as described in Example 1.

Each mutant colony was inoculated in 200 μl Minimal Media and incubated with shaking at 37° C. for 20 hours. 10 μl of the culture was then added to 190 μl of fresh Minimal Media and incubated again with shaking at 37° C. for 20 hours. 10 μl of the second culture was then added to 190 μl Auto-Induction Media and incubated with shaking at 25° C. for 18 hours to allow expression of the non-luminescent peptide mutant.

To assay each mutant culture, 30 μl of assay lysis buffer (50 mM HEPES pH 7.5, 0.3× Passive Lysis Buffer (Promega Corporation)) and 0.006 U/μl RQ1 DNase (Promega Corporation)) containing non-luminescent polypeptide (1:10 dilution of wild-type non-luminescent polypeptide E. coli clarified lysate) was added. The samples were shaken at room temperature for 10 minutes, and then 50 μl NANOGLO Luciferase Assay Reagent (Promega Corporation) added. The samples were incubated at room temperature for 10 minutes, and luminescence was measured on a GLOMAX luminometer with 0.5 s integrations.

FIG. 1 shows the luminescence (RLUs) detected in each non-luminescent peptide mutant. The results demonstrate various positions that are able to tolerate a mutation without substantial loss in luminescence, as well as a few specific mutations that improve luminescence.

Example 8 Effect of Orientation of Fusion Tag on Luminescence

In the following example, luminescence generated by non-luminescent peptides with N- or C-terminus HaloTag protein was compared.

Single colony of each peptide-HT fusion was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example 7. FIGS. 6 and 7 demonstrate the luminescence (RLUs) detected in each peptide-HT fusion. The results demonstrate combinations of mutations that produce similar luminescence as NLpep1.

Example 9 Effect of Multiple Freeze-Thaw Cycles on non-luminescent peptides

1 ml of NLpep9-HT was frozen on dry ice for 5 minutes and then thawed in a room temperature water bath for 5 minutes. 60 μl was then removed for assaying. The freeze-thaw procedure was then repeated another 10 times. After each freeze-thaw cycle, 60 μl of sample was removed for assaying.

To assay, 20 μl of each freeze-thaw sample was mixed with 30 μl of SEQ ID NO:2 and incubated at room temperature for 10 minutes. 50 μl of NANOGLO Luciferase Assay Reagent was added, and the samples incubated at room temperature for 10 minutes. Luminescence was measured on a GLOMAX luminometer with 0.5 s integrations. The results are depicted in FIG. 8 and demonstrate that NLpep can be subjected to multiple freeze-thaw cycles without a loss in activity (luminescence).

Example 10 Distinction of Mutations in Non-Luminescent Peptides

In the following example, TMR gel analysis was used to normalize the concentration of the non-luminescent peptide mutants to distinguish mutations that alter the expression from those that alter luminescence (e.g., altered luminescence may stem from altered binding affinity).

5 ml of LB was inoculated with a single mutant peptide colony and incubated with shaking at 37° C. for 20 hours. 50 μl of the overnight culture was diluted into 5 ml of fresh LB and incubated with shaking at 37° C. for 3 hours. 50 μl of 20% rhamnose was then added and incubated with shaking at 25° C. for 18 hours.

For TMR gel analysis, 79 μl of each induced culture was mixed with 10 μl 10× Fast Break Lysis Buffer (Promega Corporation), 10 μl of a 1:100 dilution of HALOTAG TMR ligand (Promega Corporation) non-luminescent polypeptide and 10 μl of RQ1 DNase and incubated at room temperature for 10 minutes. 33.3 μl of 4×SDS-loading buffer was added, and the samples incubated at 95° C. for 5 minutes. 15 μl of each sample was loaded onto an SDS gel and run according to the manufacturer's directions. The gel was then scanned on a Typhoon. Each culture was diluted based on the TMR-gel intensity to normalize concentrations. 20 μl of each diluted culture was then mixed with 30 μl assay lysis buffer containing non-luminescent polypeptide (1:10 dilution of SEQ ID NO: 2 E. coli clarified lysate) and incubated with shaking at room temperature for 10 minutes. 50 μl of NANOGLO Luciferase Assay Reagent was added, and the samples incubated at room temperature for 10 minutes. Luminescence was measured on a GLOMAX luminometer with 0.5 s integrations (SEE FIG. 9).

Example 11 Site Saturation in Non-Luminescent Polypeptide

In the following example, positions 11, 15, 18, 31, 58, 67, 106, 149, and 157 were identified as sites of interest from screening the library of random mutations in wild-type non-luminescent polypeptide. All 20 amino acids at these positions (built on 5A2 non-luminescent mutant generated in Example 6 (SEQ ID NOS: 539 and 540) to validate with other mutations in the 5A2 mutant) were compared to determine the optimal amino acid at that position. Mutant non-luminescent polypeptides were generated as previously described in Example 1. Single colony of each non-luminescent polypeptide mutant was grown according to the procedure used in Example 6. The bacterial cultures were also induced according to the procedure used in Example 6. Luminescence was assayed and detected according to the procedure used in Example 6 expect NLpep53 E. coli clarified lysate was used at 1:11.85 dilution. FIGS. 10-18 demonstrate the effect of the mutations on the ability to produce luminescence with and without NLpep.

Example 12 Comparison of Cysteine Vs. Proline as First Amino Acid in Non-Luminescent Peptide

In the following example, a comparison of using cysteine or proline as first amino acid (after necessary methionine) in the non-luminescent peptide was performed. The mutant non-luminescent peptides were generated as previously described in Example 1. Single colony of each non-luminescent polypeptide mutant was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example 7. FIG. 19 demonstrates that both cysteine and proline can be used as the first amino acid of NLpep and produce luminescence.

Example 13 Identification of the Optimal Composite Set of Mutations for the Non-Luminescent Peptide

In the following examples, an optimal composite set(s) of mutations for the non-luminescent peptide were identified. The mutant non-luminescent peptides were generated as previously described in Example 1.

-   -   1) For non-luminescent peptide composite mutants NLpep53,         NLpep66, NLpep67, and NLpep68, a single colony of each was grown         according to the procedure used in Example 10. The bacterial         cultures were also induced according to the procedure used in         Example 10. TMR gel analysis and luminescence was assayed and         detected according to the procedure used in Example 10. The         results in FIG. 20 demonstrate the luminescence as well as         the E. coli expression of NLpeps containing multiple mutations.     -   2) For non-luminescent peptide composite mutants NLpep53 and         NLpeps 66-74, a single colony of each was grown according to the         procedure used in Example 7. The bacterial cultures were also         induced according to the procedure used in Example 7.         Luminescence was assayed and detected according to the procedure         used in Example 7. The results in FIG. 21 demonstrate the         luminescence of NLpeps containing multiple mutations.     -   3) For non-luminescent peptide     -   composite mutants NLpep53 and NLpeps 66-76, a single colony of         each was grown according to the procedure used in Example 7. The         bacterial cultures were also induced according to the procedure         used in Example 7 Luminescence was assayed and detected         according to the procedure used in Example 7 except the         non-luminescent polypeptide was 5A2 or 5A2+R11E (1:10 dilution         of E. coli clarified lysate). The results in FIG. 22 demonstrate         the luminescence of NLpeps containing multiple mutations with         5A2 or 5A2+R11E. These results also demonstrate the lower         luminescence when the NLpoly mutation R11E is complemented with         an NLpep containing E as the 9th residue (NLpep72, 75, and 76).     -   4) For non-luminescent peptide composite mutants NLpep1,         NLpep69, NLpep78 and NLpep79, a single colony of each was grown         according to the procedure used in Example 7. The bacterial         cultures were also induced according to the procedure used in         Example 7. Luminescence was assayed and detected according to         the procedure used in Example 7 except the non-luminescent         polypeptide was WT (1:10 dilution of E. coli clarified lysate).         The results in FIG. 23 demonstrate the luminescence of NLpeps         containing multiple mutations.

Example 14 Composite Non-Luminescent Polypeptide Mutants

In the following example, 9 mutations from the library screens were combined into a composite clone (NLpoly1, SEQ ID NOS: 941,942), and then one of the mutations reverted back to the original amino acid (NLpoly2-10, SEQ ID NOS: 943-960) in order to identify the optimal composite set. Based on previous results of NLpoly1-10, NLpoly11-13 (SEQ ID NOS: 961-966) were designed and tested for the same purpose. Mutant NLpolys were generated as previously described in Example 1. Single colony of each non-luminescent polypeptide mutant was grown according to the procedure used in Example 6. The bacterial cultures were also induced according to the procedure used in Example 6 Luminescence was assayed and detected according to the procedure used in Example 6 expect NLpep53 E. coli clarified lysate was used at 1:11.85 dilution.

FIG. 24 demonstrates the luminescence of NLpolys containing multiple mutations.

Example 15 Substrate Specificity of Non-Luminescent Polypeptide Mutants

The following example investigates the substrate specificity of the non-luminescent polypeptide mutants. Luminescence generated from luminescent complexes formed from various non-luminescent polypeptide mutants, either Furimazine or coelenterazine as a substrate, and various non-luminescent peptides.

HEK 293 cells were plated at 100,000 cells/ml into wells of a 24 well plates containing 0.5 ml DMEM+10% FBS (50,000/well). The cells were incubated in a 37° C., 5% CO₂ incubator overnight. DNA for expression of each non-luminescent polypeptide mutant was transfected in duplicate. 1 ug plasmid DNA containing a non-luminescent polypeptide mutant was mixed with OptiMEM (Life Technologies) to a final volume of 52 ul. 3.3 μl of Fugene HD (Promega Corporation) was added, and samples incubated for 15 minutes at room temperature. 25 μl of each sample mixture was added to two wells and incubated overnight in a 37° C., 5% CO₂ incubator overnight. After overnight incubation, the growth media was removed and 0.5 ml DMEM (without phenol red)+0.1% Prionex added. The cells were then frozen on dry ice (for how long) and thawed prior to detecting luminescence.

In FIGS. 25-26, luminescence was assayed and detected according to the procedure used in Example 6, except NLpep53 E. coli clarified lysate was used at 1:10 dilution and either Furimazine or coelenterazine in either NanoGlo Luciferase Assay buffer or DMEM were used. This data demonstrates the luminescence of NLpolys in NANOGLO and DMEM with either Furimazine or Coelenterazine as the substrate. This indicates the substrate specificity (Furimazine versus Coelenterazine) of the NLpoly in both NANOGLO and DMEM.

In FIG. 27, luminescence was assayed and detected according to the procedure used in Example 6, except E. coli clarified lysate from various non-luminescent peptides (NLpep1, NLpep9, NLpep48, NLpep53, NLpep69 or NLpep76) were used at 1:10 dilution. In addition, either Furimazine or coelenterazine in either NanoGlo Luciferase Assay buffer were used. This data demonstrates the substrate specificity of NLpoly/NLpep pairs.

In FIG. 28, luminescence was assayed and detected by separately diluting NLpep53-HT fusion 1:10 and the non-luminescent polypeptide lysates 1:10 in DMEM+0.1% Prionex. 20 μl of non-luminescent peptide and 20 μl non-luminescent polypeptide were then combined and incubated for 10 minutes at room temperature. 40 μl of NanoGlo Buffer with 100 uM Furimazine or DMEM with 0.1% Prionex and 20 uM Furimazine was then added to the samples, and luminescence detected on GloMax Multi. This data demonstrates the substrate specificity of NLpolys expressed in HEK293 cells.

In FIG. 29, luminescence was assayed and detected by separately diluting NLpep1-HT, NLpep53-HT, NLpep69-HT or NLpep76-HT fusion 1:10 and the non-luminescent polypeptide lysates 1:10 in DMEM+0.1% Prionex. 20 μl of non-luminescent peptide and 20 μl non-luminescent polypeptide were then combined and incubated for 10 minutes at room temperature. 40 μl of NanoGlo Buffer with 100 uM Furimazine or DMEM with 0.1% Prionex and 20 uM Furimazine was then added to the samples, and luminescence detected on GloMax Multi. This data demonstrates the luminescence of NLpolys expressed in mammalian cells and assayed with various NLpeps.

Example 16 Signal-to-Background of Non-Luminescent Polypeptide Mutants with Furimazine or Coelenterazine

The following example investigates signal-to-background of the non-luminescent polypeptide mutants. Luminescence generated from various non-luminescent polypeptide mutants was measured using either Furimazine or coelenterazine as a substrate as well as with various non-luminescent peptides.

HEK 293 cells were plated at 15,000 cells/well in 100 μl DMEM+10% FBS into wells of 96-well plates. The cells were incubated in a 37° C., 5% CO₂ incubator overnight. Transfection complexes were prepared by adding 0.66 ug each of plasmid DNA for expression of a non-luminescent polypeptide mutant and a non-luminescent peptide mutant plasmid to a final volume of 31 μl in OptiMem. 2 μl Fugene HD was added to each transfection complex and incubated for 15 minutes at room temperature. For each peptide/polypeptide combination, 5 μl of a transfection complex was added to 6 wells of the 96-well plate and grown overnight at 37 C in CO₂ incubator. After overnight incubation, the growth media was removed and replaced with CO₂-independent media containing either 20 uM coelenterazine or 20 uM Furimazine. The samples were incubated for 10 minutes at 37° C., and kinetics measured over the course of 1 hour at 37° C. on a GloMax Multi+. FIG. 30 demonstrates the substrate specificity of various NLpoly/NLpep pairs when the NLpoly is expressed in mammalian cells.

Example 17 Luminescence and Substrate Specificity

The following example investigates the luminescence and substrate specificity of various non-luminescent polypeptide mutants with NLpep69 and using either Furimazine or coelenterazine as a substrate.

CHO cells were plated at 20,000 cells/well in 100 μl of DMEM+10% FBS into wells of 96-well plates. The cells were incubated in a 37° C., 5% CO2 incubator overnight. Transfection complexes were prepared by adding 0.66 ug each of plasmid DNA for expression of a non-luminescent polypeptide mutant and a non-luminescent peptide mutant plasmid to a final volume of 31 μl in OptiMem. 2 μl Fugene HD was added to each transfection complex and incubated for 15 minutes at room temperature. For each peptide/polypeptide combination, 5 μl of transfection complex was added to 6 wells of the 96-well plate and grown overnight at 37 C in CO₂ incubator. After overnight incubation, the growth media was removed and replaced with CO₂— independent media containing either 20 uM coelenterazine or 20 uM Furimazine. The samples were incubated for 10 minutes at 37° C., and kinetics measured over the course of 1 hour at 37° C. on a GloMax Multi+. FIG. 31 demonstrates the substrate specificity when NLpolys are coexpressed in mammalian cells with NLpep69.

Example 18 Luminescence and Substrate Specificity Between Live-Cell and Lytic Conditions

The following example investigates the luminescence and substrate specificity of various non-luminescent polypeptide mutants with NLpep69, NLpep78 or NLpep79, using either Furimazine or coelenterazine as a substrate and under either lytic or live cell conditions.

HEK 293 cells were plated at 15,000 cells/well in 100 μl DMEM+10% FBS into wells of 96-well plates. The cells were incubated in a 37° C., 5% CO2 incubator overnight. Transfection complexes were prepared by adding 0.66 ug each of plasmid DNA for expression of a non-luminescent polypeptide mutant and a non-luminescent peptide mutant plasmid to a final volume of 31 μl in OptiMem. 2 μl Fugene HD was added to each transfection complex and incubated for 15 minutes at room temperature. For each NLpoly-NLpep combination, 5 μl of transfection complex was added to 6 wells of the 96-well plate and grown overnight at 37 C in CO2 incubator. After overnight incubation, the growth media was removed and replaced with CO2- independent media containing either 20 uM coelenterazine or 20 uM Furimazine. The samples were incubated for 10 minutes at 37° C., and kinetics measured over the course of 1 hour at 37° C. on a GloMax Multi+. FIGS. 32-34 demonstrate the substrate specificity of NLPolys coexpressed in mammalian cells with NLpep69, 78, or 79 in live-cell and lytic formats.

Example 19 Comparison of Non-Luminescent Polypeptide Mutants Expressed in E. coli

A single colony of each non-luminescent polypeptide was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example 7 except NLpep78-HT or NLpep79-HT at 1:1,000 dilution was used. FIG. 35 demonstrates the luminescence of NLpolys expressed in E. coli and assayed with NLpep78 or 79.

Example 20 Ability of non-luminescent polypeptide clones to produce luminescence without complementing non-luminescent peptide

A single colony of each non-luminescent polypeptide was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example 7 except no non-luminescent peptide was added to the assay buffer. FIG. 36 demonstrates the luminescence of NLpolys expressed in E. coli and assayed in the absence of NLpep.

Example 21 Substrate Specificity of Non-Luminescent Polypeptide Mutants Expressed in E. coli

A single colony of each non-luminescent polypeptide was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example except either Furimazine or coelenterazine was mixed with NANOGLO Assay Buffer. FIG. 37 demonstrates the substrate specificity of NLpolys expressed in E. coli and assayed with NLpep78 or 79.

Example 22 Improved Luminescence of Non-Luminescent Polypeptide Mutants with NLpep78

Complementation of the non-luminescent polypeptide mutants with NLpep78-HT was demonstrated in CHO and Hela cells.

CHO and Hela cells (CHO: 100,000 seeded the day prior to transfection; Hela: 50,000 seeded the day prior to transfection) were transfected with 5 ng of a non-luminescent polypeptide mutant 5A2 or 5P or with wild-type non-luminescent polypeptide using Fugene HD into wells of a 24-well plate and incubated at 37° C. overnight. After the overnight incubation, the media was replaced with DMEM without phenol red, and the cells frozen at −80° C. for 30 minutes. The cells were then thawed and transferred to a 1.5 ml tube. The cell lysates were then diluted 1:10 DMEM without phenol red, 20 μl mixed with NLpep78 (NLpep78-HT7 E. coli lysate diluted 1:1,000 in DMEM without phenol red) and shaken at room temperature for 10 minutes. 40 μl DMEM without phenol red and 20 uM Furimazine was added and luminescence measured on a GloMax with a 0.5 second integration. FIG. 38 demonstrates the luminescence of NLpolys expressed in mammalian cells and assayed with NLpep78.

Example 23 Non-Luminescent Polypeptide Fusions and Normalizing Non-Luminescent Polypeptide Concentrations

A comparison of raw and normalized luminescence from non-luminescent polypeptide fused to either firefly luciferase (FIG. 39) or click beetle red luciferase (FIG. 40) were performed to provide insight into how much benefit, e.g., in expression, solubility and/or stability, stems from the concentration of the non-luminescent polypeptide as well as complementation as a fusion non-luminescent polypeptide.

HEK293, Hela or CHO cells were transfected with 5 ng 5P NLpoly-firefly luciferase fusion, 5P NLpoly-click beetle luciferase fusion, wild-type 5P-firefly luciferase fusion or wild-type 5P-click beetle luciferase fusion according to the procedure in Example 22. Lysates were also prepared according to Example 22. The cell lysates were then diluted 1:10 DMEM without phenol red, 20 μl mixed with NLpep78 (diluted 1:100 in DMEM without phenol red; E. coli lysate) and shaken at room temperature for 10 minutes. 40 μl NanoGlo with 20 uM Furimazine or Bright-Glo (Promega Corporation) was added and luminescence measured on a GloMax with 0.5 second integration. FIGS. 39 and 40 demonstrate the specific activity of 5P versus WT NLpoly expressed in mammalian cells and assayed with NLpep78.

Example 24 Complementation in Live Cells

This example demonstrates complementation in live-cells using either wild-type or 5P NLpoly. Hela cells plated into wells of 96-well plated, transfected with 0.5 ng of wild-type or 5P non-luminescent polypeptide plasmid DNA using Fugene HD and incubated at 37° C. overnight. After the overnight incubation, the cells were then transfected with 0.5 ng NLpep78-HT plasmid DNA using Fugene HD and incubated at 37° C. for 3 hours. The media was then replaced with CO₂-independent media+0.1% FBS and 20 uM PBI-4377, and luminescence measured at 37° C. on a GloMax with 0.5 second integration. FIG. 41 demonstrates the live-cell complementation between 5P or WT NLpoly and NLpep78.

Example 25 Complementation in Cell-Free Extract

To demonstrate complementation in cell-free extract, 0.5 ug NLpep78-HT and 0.5 ug non-luminescent polypeptide mutant plasmid DNA were mixed with TNT rabbit reticulocyte lysate master mix (Promega Corporation) and incubated at 30° C. for 1 hour. 25 μl of the cell-free expression extract was mixed with 25 μl NanoGlo Luciferase Assay reagent and incubated at room temperature for 10 minutes. Luminescence was measured on a GloMax with 0.5 second integration. FIG. 42 demonstrates luminescence from complementing NLpoly/NLpep pairs expressed in a cell-free format.

Example 26 Binding Affinity of Non-Luminescent Polypeptide Expressed in Mammalian Cells with Synthetic Non-Luminescent Peptide

To demonstrate the binding affinity between non-luminescent polypeptide and non-luminescent peptide pairs, non-luminescent polypeptide lysates from Hela, HEK293 and CHO cells were prepared as previously described and diluted 1:10 PBS+0.1% Prionex. 4× concentrations of non-luminescent peptide (synthetic) were made in PBS+0.1% Prionex. 20 μl of the non-luminescent polypeptide lysate was mixed with 20 μl non-luminescent peptide and shaken at room temperature for 10 minutes. 40 μl of NanoGlo Luciferase Assay Reagent or PBS+0.1% Prionex with Furimazine was added and shaken at room temperature for 10 minutes. Luminescence was detected on a GloMax with 0.5 s integration. Kd values were determined using Graphpad Prism, One Site-Specific Binding. FIGS. 43 and 44 demonstrate the dissociation constants measured under various buffer conditions (PBS for complementation then NanoGlo for detection, PBS for complementation and detection, NanoGlo for complementation and detection).

Example 27 Improved Binding Affinity when Cysteine Mutated to Phenylalanine in Non-Luminescent Peptide Mutants

To demonstrate improved binding affinity in non-luminescent peptide mutants with a mutated cysteine at the 8^(th) residue of the peptide, non-luminescent polypeptide mutant lysates from Hela, HEK293 and CHO cells were prepared as previously described and diluted 1:10 PBS+0.1% Prionex. 4× concentrations of non-luminescent peptide (NLpep) were made in PBS+0.1% Prionex+10 mM DTT. 20 μl of the non-luminescent polypeptide lysate was mixed with 20 μl non-luminescent peptide and shaken at room temperature for 10 minutes. 40 μl of NanoGlo Luciferase Assay Reagent was added and shaken at room temperature for 10 minutes. Luminescence was detected on a GloMax with 0.5 s integration. FIG. 45 demonstrates NLpep C8F mutation significantly improves the binding affinity for 5P.

Example 28 Detectable Luminescence of Polypeptide Variants without Non-Luminescent Peptide in Hela Cells

To demonstrate luminescence in non-luminescent polypeptide without non-luminescent peptide, Hela cells (10,000 seeded the day prior to transfection) in wells of a 96-well plate were transfected with varying amounts of non-luminescent polypeptide+pGEM-3zf Carrier DNA to a total of 50 ng using Fugene HD and incubated 37° C. overnight. After incubation, the media was replaced with CO₂-independent media+0.1% FBS+20 uM Furimazine and incubated at 37° C. for 10 minutes, and luminescence detected on a GloMax with 0.5 s integration. FIG. 46 demonstrates the luminescence of NLpoly WT or 5P in live Hela cells without NLpep after transfection of various amounts of plasmid DNA.

Example 29 Generation of Additional Non-Luminescent Polypeptide Variants

Additional non-luminescent polypeptide variants: Ile-11 (Ile at residue 11), Val-11, Tyr-11, Glu-11, Glu-157, Pro-157, Asp-157, Ser-157, Met-149, Leu-106, NLpoly11, and NLpoly12 were generated as described below, and their expression analyzed. The additional non-luminescent polypeptide variants were made in the 5A2 non-luminescent polypeptide background.

Fresh individual colonies (KRX) of each additional non-luminescent polypeptide variants were picked and grown overnight in LB+ampicillin (100 ug/ml) at 30° C. and then diluted 1:100 in LB+ampicillin and grown at 37° C. for 2.5 hours (OD₆₀₀ ˜0.5). Rhamnose was added to a final concentration of 0.2%, and the cells were split in triplicate and grown overnight at 25° C. for ˜18 h. Cells were lysed using 0.5× Fast Break for 30 minutes at ambient temperature, snap-frozen on dry ice, and stored at −20° C. Upon fast thawing, soluble fractions were prepared by centrifugation at 10K for 15 min at 4° C. Samples were assayed for luminescence on a Tecan Infinite F-500 luminometer.

FIG. 49 demonstrates that total lysate and soluble fraction of each non-luminescent polypeptide variant as analyzed by SDS-PAGE. The data provides information about expression, solubility and stability of the additional non-luminescent polypeptide variants. A majority of the additional non-luminescent polypeptide variants produced more protein (total and soluble) than wild-type, but in many cases, the difference is subtle. Improved expression for NLpoly11 and NLpoly12 was more noticeable.

Example 30 Background Luminescence of Additional Non-Luminescent Polypeptide Variants

The background luminescence of the additional non-luminescent polypeptide variants generated in Example 29 was measured by incubating 25 μl of non-luminescent polypeptide variant lysate with 25 μl DMEM at room temperature for 10 minutes. 50 μl NanoGlo Luciferase Assay Reagent was then added, and luminescence measured at 5 and 30 minutes on a Tecan Infinite F500. NLpep53 (Pep 53) alone and DMEM (DMEM) alone were used as controls. FIG. 47 demonstrates that a majority of the additional non-luminescent polypeptide variants showed elevated background luminescence.

Example 31 Luminescence of Additional Non-Luminescent Polypeptide Variants after Complementation

Luminescence of the additional non-luminescent polypeptide variants generated in Example 28 was measured by incubating 25 μl of non-luminescent polypeptide variant lysate with 25 μl NLpep-53 at room temperature for 10 minutes 50 μl NanoGlo Luciferase Assay Reagent was then added, and luminescence measured at 5 and 30 minutes on a Tecan Infinite F500. NLpep53 (Pep 53) alone and DMEM (DMEM) alone were used as controls. FIG. 48 demonstrates that the non-luminescent polypeptide variants Val-11, Glu-11, Glu-157, Pro-157, Asp-157, Ser-157 and Met-149 generated significantly more luminescence than parental 5A2.

Example 32 Correlation Between Increased Background Luminescence of Non-Luminescent Polypeptide in the Absence of Non-Luminescent Peptide and Amount of Protein in Soluble Fraction

Individual colonies of the non-luminescent polypeptide variants 3P, 3E, 5P, 5E, 6P and 6E were picked and grown overnight in LB+ampicillin at 30° C. and then diluted 1:100 in LB+ampicillin and grown at 37° C. for 2.5 hours (OD₆₀₀ ˜0.5). Rhamnose was added to a final concentration of 0.2%, and the cells were split in triplicate and grown overnight at 25° C. for ˜18 h. Cells were lysed using 0.5× Fast Break for 30 minutes at ambient temperature, snap-frozen on dry ice, and stored at −20° C. Upon fast thawing, soluble fractions were prepared by centrifugation at 10K for 15 min at 4° C. Samples were assayed for luminescence on a Tecan Infinite F-500. FIG. 50A shows the total lysate and soluble fraction of each non-luminescent polypeptide variant. FIG. 50B shows the background luminescence of each non-luminescent polypeptide variant. FIG. 51 shows the luminescence generated with each non-luminescent polypeptide variant when complemented with 10 or 100 nM NLpep78 (NVSGWRLFKKISN) in LB medium.

Example 33 Elongations and Deletions of Non-Luminescent Polypeptide

The non-luminescent polypeptide variant 5P was either elongated at the C-terminus by the addition of the residues VAT, AA, VTG, VT, VTGWR, VTGW, V, A, VA, GG, AT, GTA, ATG or GT or deletion of 1 to 7 residues at the C-terminus of 5P, e.g., D1=deletion of 1 residue, D2=deletion of 2 residues, etc. Background luminescence in E. coli lysates (FIG. 52) and luminescence generated after complementation with NLpep78 (FIG. 53; NVSGWRLFKKISN) or NLpep79 (FIG. 54; NVTGYRLFKKISN) were measured. FIG. 55 shows the signal-to-background of the non-luminescent polypeptide 5P variants. FIG. 56 provides a summary of the luminescent results. FIG. 57 shows the amount of total lysate and soluble fraction in each non-luminescent polypeptide 5P variant.

Example 34 Comparison of 5P and I107L Non-Luminescent Polypeptide Variant

FIG. 58 shows the amount of total lysate and soluble fraction of 5P and I107L (A), luminescence generated by 5P or I107L without non-luminescent peptide or with NLpep78 or NLpep79 (B) and the improved signal-to-background of I107L over 5P(C).

Example 35 Generation of 5P Non-Luminescent Polypeptide Mutants

Mutations identified in a screening of random mutations in the 5P non-luminescent polypeptide variant were generated as previously described. Each single 5P non-luminescent polypeptide mutant colony was inoculated in 200 μl Minimal Media and incubated with shaking at 37° C. for 20 hours. 10 μl of the culture was then added to 190 μl of fresh Minimal Media and incubated again with shaking at 37° C. for 20 hours. 10 μl of the second culture was then added to 190 μl Auto-Induction Media (Minimal Media+5% glucose+2% rhamnose) and incubated with shaking at 25° C. for 18 hours to allow expression of the non-luminescent polypeptide mutant. 10 μl of the 5P non-luminescent polypeptide mutant expression culture was added to 40 μl of assay lysis buffer containing NLpep78-HT (1:386 dilution) or NLpep79-HT (1:1,000 dilution) and shaken at room temperature for 10 minutes. 50 μl of NanoGlo Assay Buffer containing 100 uM coelenterazine was added and shaken at room temperature for 10 minutes. Luminescence was measured on GloMax with 0.5 sec integration. FIGS. 59-62A shows background luminescence while FIGS. 59-62B and C show luminescence generated after complementation with NLpep78 or NLpep79.

Example 36 Binding Affinity Between Elongated Non-Luminescent Polypeptide Variant and Deleted Non-Luminescent Peptide

The binding affinity between an elongated non-luminescent polypeptide variant, i.e., containing additional amino acids at the C-terminus, and a deleted non-luminescent peptide, i.e., deleted amino acids at the N-terminus.

Lysates of E. coli expressing non-luminescent polypeptide 5P/+V/+VT/+VTG prepared as previously described were diluted 1:2000 in PBS+0.1% Prionex. 25 μl of the diluted lysate was incubated with 25 μl of NLpep78, NLpep80, NLpep81 or NLpep82 (diluted 0-500 nM in dilution buffer) for 5 min at room temp. 50 μl of Furimazine diluted to 1× with NanoGlo Assay Buffer was added to each sample and incubated for 10 minutes at room temperature. Luminescence was measured on a GloMax Multi with 0.5 s integration time. FIG. 63 demonstrates the binding affinity between NLpolys with additional amino acids at the C-terminus with NLpeps with amino acids deleted from the N-terminus

Example 37 Binding Affinity Between Non-Luminescent Polypeptide Expressed in E. coli and Synthetic Non-Luminescent Peptide

Non-luminescent polypeptide LB lysates were prepared and diluted 1:100 into PBS+0.1% Prionex. 2× dilutions of synthetic NLpep78 were made in PBS+0.1% Prionex. 25 μl of the diluted non-luminescent polypeptide lysate was mixed with 25 μl of each dilution of non-luminescent peptide and incubated 3 minutes at ambient temperature. 50 μl of NanoGlo Luciferase Assay Reagent was added, incubated for 5 minutes at room temperature, and luminescence measured on a GloMax Multi+. FIG. 64 shows the calculated Kd values using one-site specific binding.

Example 38 Binding Affinity Between 5P Non-Luminescent Polypeptide Expressed in Mammalian Cells and NLpep80 or NLpep87

Lysates of CHO, HEK293T, or HeLa cells expressing NLpoly 5P were diluted 1:1000 in dilution buffer (PBS+0.1% Prionex.) 25 μl of diluted lysate was incubated with 25 μl of NLpep80/87 (diluted 0-5 μM in dilution buffer) for 5 min at room temp. 50 μl of furimazine (diluted to 1× with NanoGlo buffer) was added to each well, and the plate was incubated for 10 min at room temp. Luminescence was then read on a GloMax Multi with 0.5 s integration time (FIG. 65).

Example 39 Binding Affinity Between 5P Non-Luminescent Polypeptide Expressed in E. coli and NLpep80 or NLpep87

Lysates of E. coli expressing NLpoly 5P were diluted 1:2000 in dilution buffer (PBS+0.1% Prionex.) 25 μl of diluted lysate was incubated with 25 μl of NLpep80/87 (diluted 0-5 μM in dilution buffer) for 5 min at room temp. 50 μl of furimazine (diluted to 1× with NanoGlo buffer) was added to each well, and the plate was incubated for 10 min at room temp. Luminescence was then read on a GloMax Multi with 0.5 s integration time (FIG. 66).

Example 40 Complementation Between a Deleted Non-Luminescent Polypeptide and Elongated Non-Luminescent Peptide

Complementation between a deleted non-luminescent polypeptide, i.e., amino acids deleted from the C-terminus, and an elongated non-luminescent peptide, i.e., amino acids added to the N-terminus, was performed. NLpep-HT E. coli clarified lysates as prepared as previously described in Example 6. The amount of NLpep-HT was quantitated via the HaloTag fusion. Briefly, 10 μl of clarified lysate was mixed with 10 μl HaloTag-TMR ligand (diluted 1:100) and 80 μl water and incubated at room temperature for 10 minutes. 33.3 μl 4×SDS Loading Buffer was added and incubated at 95° C. for 5 minutes. 15 μl was loaded onto an SDS-PAGE gel and imaged on a Typhoon. Based on the intensities from the SDS-PAGE gel, non-luminescent peptides were diluted in PBS+0.1% Prionex non-luminescent peptides to make equivalent concentrations. The non-luminescent polypeptide lysates were then diluted 1:100 in PBS+0.1% Prionex. 20 μl of diluted non-luminescent polypeptide and 20 μl diluted non-luminescent peptide were mixed and shaken at room temperature for 10 minutes. 40 μl NanoGlo Luciferase Assay Reagent was added and shaken at room temperature for 10 minutes. Luminescence was measured on a GloMax using 0.5 sec integration. FIG. 67 demonstrates the luminescence of NLpolys with amino acids removed from the C-terminus with NLpeps with additional amino acids on the N-terminus

Example 41 Binding Affinity Between 5P Non-Luminescent Polypeptide Expressed in Hela Cells and NLpep78 or Truncated NLpep78 (NLpep80-87)

5P non-luminescent polypeptide lysate was prepared from Hela cells as previously described and diluted prepared 1:10 in PBS+0.1% Prionex. 4× concentrations (range determined in preliminary titration experiment) of non-luminescent peptide (synthetic peptide; by Peptide 2.0 (Virginia); made at either 5, 10, or 20 mg scale; blocked at the ends by acetylation and amidation, and verified by net peptide content analysis) was prepared in PBS+0.1% Prionex. 20 μl P non-luminescent polypeptide and 20 μl non-luminescent peptide were mixed and shaken at room temperature for 10 minutes. 40 μl of NanoGlo Luciferase Assay reagent was added and shaken at room temperature for 10 minutes. Luminescence was measured on GloMax with 0.5 s integration. FIG. 68 demonstrates the binding affinity and corresponding luminescence between 5P and truncated versions of NLpep78. The binding affinity is increased when 1 amino acid is removed from the N-terminus, the C-terminus, or 1 amino acid from each terminus. Removing more than 1 amino acid from either terminus lowers the affinity but does not always lower the Vmax to the same extent.

Example 42 Binding Affinity Between Elongated Non-Luminescent Polypeptide and Truncated Non-Luminescent Peptide

The binding affinity between an elongated non-luminescent polypeptide, i.e., one with 2 extra amino acids on C-terminus, and a truncated non-luminescent peptide, i.e., one with 2 amino acids removed from N-terminus (NLpep81), was determined.

Non-luminescent polypeptide lysate was prepared as previously described and diluted prepared 1:100 in PBS+0.1% Prionex. 2× dilutions of NLpep81 (synthetic peptide; by Peptide 2.0 (Virginia); made at either 5, 10, or 20 mg scale; blocked at the ends by acetylation and amidation, and verified by net peptide content analysis) was prepared in PBS+0.1% Prionex. 25 μl non-luminescent polypeptide and 25 μl of each non-luminescent peptide dilution were mixed and shaken at room temperature for 3 minutes. 50 μl of NanoGlo Luciferase Assay reagent was added and shaken at room temperature for 5 minutes. Luminescence was measured on GloMax with 0.5 s integration. FIG. 69 shows the calculate Kd values using one-site specific binding.

Example 43 Binding Affinity Between Elongated Non-Luminescent Polypeptide and Truncated Non-Luminescent Peptide

The binding affinity between an elongated non-luminescent polypeptide, i.e., one with 3 extra amino acids on C-terminus, and a truncated non-luminescent peptide, i.e., one with 3 amino acids removed from N-terminus (NLpep82), was determined.

Non-luminescent polypeptide lysate was prepared and diluted prepared 1:100 in PBS+0.1% Prionex. 2× dilutions of NLpep82 (synthetic peptide; by Peptide 2.0 (Virginia); made at either 5, 10, or 20 mg scale; blocked at the ends by acetylation and amidation, and verified by net peptide content analysis) was prepared in PBS+0.1% Prionex. 25 μl non-luminescent polypeptide and 25 μl of each non-luminescent peptide dilution were mixed and shaken at room temperature for 3 minutes. 50 μl of NanoGlo Luciferase Assay reagent was added and shaken at room temperature for 5 minutes. Luminescence was measured on GloMax with 0.5 s integration. FIG. 70 shows the calculate Kd values derived using one-site specific binding.

Example 44 Binding Affinity Between Non-Luminescent Polypeptide Clones Expressed in E. coli and Synthetic NLpep78

Non-luminescent polypeptide variants were grown in M9 minimal media. Individual colonies were inoculated and grown overnight at 37° C. Samples were diluted 1:20 in M9 minimal media and grown overnight at 37° C. Samples were again diluted 1:20 in M9 induction media and grown overnight at 25° C. Samples were pooled, and 100 μl of the pooled cells were lysed with 400 μl of PLB lysis buffer and incubate at room temperature for 10 minutes. The lysates were diluted 1:100 in PBS+0.1% Prionex. 2× dilutions of synthetic NLpep78 were made in PBS+0.1% Prionex. 25 μl of non-luminescent polypeptide dilution was mixed with 25 μl of each non-luminescent peptide dilution and incubated for 3 minutes at room temperature. 50 μl of NanoGlo Luciferase Assay Reagent was added, incubated at room temperature for 5 minutes, and luminescence read on GloMax Multi+. FIG. 71 shows the calculate Kd values derived using one-site specific binding.

Example 45 Determination of the Effect of Mutations on Km

Using diluted pooled lysates from Example 11, 25 μl of non-luminescent polypeptide diluted lysate (1:100 in PBS+0.1% Prionex) was mixed with 25 μl of 500 nM NLpep78 for each sample and incubated at room temperature for 5 minutes. 2× dilutions of Furimazine in NanoGlo Luciferase Assay Buffer were prepared, and 50 μl of non-luminescent peptide and non-luminescent polypeptide sample mixed with 50 μl of NanoGlo/Furimazine dilutions. Luminescence was measured after 5 minute incubation at room temperature. FIG. 72 show the calculated Km derived using Michaelis-Menten.

Example 46 Demonstration of a Three-Component Complementation

A tertiary complementation using 2 NLpeps and NLpoly 5P non-luminescent polypeptide is demonstrated. NLpoly 5P-B9 (5P with residues 147-157 deleted) and NLpep B9-HT (Met+ residues 147-157 fused to N-terminus of HT7) lysates were prepared.

a) NLpoly 5P-B9+NLpoly B9 Titration with NLpep78

NLpoly 5P-B9+NLpoly B9 was titrated with NLpep78. 20 μl 5P-B9 (undiluted) was mixed with 20 μl peptideB9-HT (undiluted). Dilutions of NLpep78 (synthetic peptide, highest concentration=100 uM) were made in PBS+0.1% Prionex. 20 μl NLpep78 was added to 40 μl of the 5P-B9+peptideB9-HT mixture and shaken at room temperature for 10 minutes. 60 μl NanoGlo Luciferase Assay Reagent was added and shaken at room temperature for 10 minutes. Luminescence was measured on GloMax with 0.5 s integration.

B) NLpoly 5P-B9+NLpep78 Titration with NLpepB9-HT

20 μl NLpoly 5P-B9 (undiluted) was mixed with 20 μl NLpep78 (100 uM). Dilutions of peptideB9-HT (highest concentration=undiluted) were made in PBS+0.1% Prionex. 20 μl of peptideB9-HT was added to 40 μl of the 5P-B9+NLpep78 mixture and shaken at room temperature for 10 minutes. 60 μl NanoGlo Luciferase Assay Reagent was added and shaken at room temperature for 10 minutes. Luminescence was measured on GloMax with 0.5 s integration.

FIG. 73 demonstrates the feasibility of a ternary system consisting of 2 different NLpeps and a truncated NLpoly. Since all 3 components are non-luminescent without the other 2, this system could be configured such that each NLpep is fused (synthetically or genetic engineering) to a binding moiety and the truncated NLpoly used at high concentrations to produce light only in the presence of an interaction between the binding moieties, or such that each of the 3 components are fused to binding moieties to produce light only in the event of ternary complex formation.

Example 47 Complementation with NLpep88 (NLpep78 with Gly as 6th Residue Instead of Arg)

NLpep88-HT and 5P E. coli clarified lysates were prepared as previously described. Serial dilutions of NLpep88-HT lysate were made in PBS+0.1% Prionex. 20 μl of 5P lysate and 20 μl NLpep88-HT lysate were mixed and shaken at room temperature for 10 minutes. 40 μl of NanoGlo Luciferase Assay Reagent was added and shaken at room temperature for 10 minutes. Luminescence was measured on GloMax with 0.5 s integration. FIG. 74 demonstrates the importance of the arginine residue at the 6th position of the NLpep. While there is no increase in luminescence above 5P alone at lower concentrations of NLpep88, high concentrations of NLpep increased the luminescence suggesting a catalytically compromised complex and not a lack of interaction between 5P and NLpep88.

Example 48 Subcellular Localization of NLpep78 and 79 as N-Terminal Fusions to HaloTag

U2OS cells were plated and left to recover overnight at 37° C. Cells were then transfected with HaloTag alone DNA construct or the HaloTag-NanoLuc peptide DNA constructs (all under the control of CMV promoter): P1-HT, P78-HT or P79-HT diluted 1:10 with carrier DNA (pSI) using FuGENE HD and incubated for 24 hours at 37° C. Cells were then labeled with HaloTag-TMR ligand by the manufacturer's standard rapid labeling protocol and imaged. FIG. 75 demonstrates that NLpep78 and 79 do not alter the intracellular localization of the HaloTag protein.

Example 49 Subcellular Localization of Non-Luminescent Polypeptide (WT and 5P)

U2OS cells were plated and left to recover overnight at 37° C. Cells were either kept as non-transfection controls or transfected with the NanoLuc DNA constructs: FL, NLpoly (wt) or NLpoly(5P) diluted 1:10 with carrier DNA (pSI) using FuGENE HD and incubated for 24 hours at room temperature. Cells were fixed and subsequently processed for ICC. ICC was done using 1:5000 GS (PRO) primary antibody overnight at 4° C. followed by an Alexa488 goat anti-rabbit secondary antibody. FIG. 76 demonstrates that both NLpoly WT and NLpoly 5P localize uniformly in cells.

Example 50 Demonstration that Non-Luminescent Polypeptide can Easily and Quickly Detect Non-Luminescent Peptide Conjugated to a Protein of Interest

99 μl of NLpep53-HT E. coli clarified lysate was mixed with 24.75 μl 4×SDS loading buffer. 1:10 serial dilutions of the lysate-loading buffer mixture were made and incubated at 95° C. for 5 minutes. 15 μl was loaded onto a SDS-PAGE gel. After gel completions, it was transferred to PVDF using iBlot and washed with 10 mL NLpoly L149M E. coli clarified lysate at room temperature for 30 minutes. The membrane was then placed on a LAS4000 imager and 2 mL NanoGlo® Luciferase Assay Reagent added. A 60 second exposure was taken (FIG. 77).

Example 51 Site Saturation at Non-Luminescent Polypeptide Positions 31, 46, 108, 144, and 157 in the Context of 5P

Single amino acid change variants were constructed onto NLpoly 5P (pF4Ag vector background) at the sites according to table 5 below. In effect, the native residue was varied to each of the 19 alternative amino acids for a total of 95 variants.

TABLE 5 Position 31 Position 46 Position 108 Position 144 Position 157 B1 Ala E3 Ala H5 Ala C8 Ala F10 Ala C1 Cys F3 Cys A6 Cys D8 Cys G10 Cys D1 Asp G3 Asp B6 Asp E8 Asp H10 Asp E1 Glu H3 Glu C6 Glu F8 Glu A11 Glu F1 Gly A4 Phe D6 Phe G8 Phe B11 Phe G1 His B4 Gly E6 Gly H8 Gly C11 Gly H1 Ile C4 His F6 His A9 His D11 His A2 Lys D4 Ile C6 Ile B9 Ile E11 Ile B2 Leu E4 Lys H6 Lys C9 Lys F11 Lys C2 Met F4 Met A7 Leu D9 Leu G11 Leu D2 Asn G4 Asn B7 Met E9 Met H11 Met E2 Pro H4 Pro C7 Pro F9 Asn A12 Asn F2 Gln A5 Gln D7 Gln G9 Pro B12 Gln G2 Arg B5 Arg E7 Arg H9 Gln C12 Arg H2 Ser C5 Ser F7 Ser A10 Arg D12 Ser A3 Thr D5 Thr G7 Thr B10 Ser E12 Thr B3 Val E5 Val H7 Val C10 Val F12 Val C3 Trp F5 Trp A8 Trp D10 Trp G12 Trp D3 Tyr G5 Tyr B8 Tyr E10 Tyr H12 Tyr

Individual colonies were grown in LB+amp and incubated overnight at 30° C. A 5P control was also included. The overnight cultures were used to inoculate fresh LB+amp (1:100), and these cultures grew for 2 hours 45 minutes at 37° C. Rhamnose was added to 0.2%, and the cultures left to grow/induce overnight at 25° C. After 18 hours of induction, cells were lysed using 0.5× FastBreak (30 min ambient temperature), snap frozen on dry ice, and stored at −20° C. Following a fast thaw, samples were assayed in the absence and presence of Pep87 (aka NLpep 87).

For the (−) peptide reactions, 30 uL lysate was incubated with 30 uL PBS pH 7.5 for 10 min and then 60 uL NanoGlo® Luciferase Assay reagent (Promega Corporation) added. After 5 minutes, luminescence was measured. For the (+) peptide reactions, 30 uL lysate was incubated with 30 uL of 8 nM Pep87. After 10 min, 60 uL NanoGlo® Luciferase Assay reagent was added, and luminescence measured at 5 minutes.

Luminescence (RLU) data for the (−) peptide samples were normalized to the readings for the 5P control, and these results are presented in FIG. 78. Luminescence (RLU) data for the (+) peptide samples were also normalized to 5P, but then also normalized to the values in FIG. 76 in order to represent signal to background (S/B; FIG. 79).

Example 52 Use of the High Affinity Between NLpoly and NLpep for Protein Purification/Pull Downs

MAGNEHALOTAG beads (Promega Corporation; G728A) were equilibrated as follows: a) 1 mL of beads were placed on magnet for ˜30 sec, and the buffer removed; b) the beads were removed from magnet, resuspended in 1 mL PBS+0.1% Prionex, and shaken for 5 min at RT; and c) steps a) and b) were repeated two more times NLpep78-HaloTag (E. coli clarified lysate) was bound to MAGNEHALOTAG beads by resuspending the beads in 1 mL NLpep78-HT clarified lysate, shaking for 1 hr at RT and placing on magnet for ˜30 sec. The lysate (flow through) was removed and saved for analysis. NLpoly 8S (E. coli clarified lysate) was bound to the NLpep78 bound-MagneHaloTag beads from the step above by resuspending the beads in 1.5 mL 8S lysate, shaking for 1 hr at RT and placing on a magnet for ˜30 sec. The lysate (flow through) was removed and saved for analysis. The beads were resuspended in 1 mL PBS+0.1% Prionex, shaken for 5 min at RT, placed on magnet for ˜30 sec, and PBS (wash) removed. The beads were washed three more times.

To elute the bound peptide/polypeptide, the beads were resuspended in 500 uL 1×SDS buffer and shaken for 5 min at RT. The beads were then placed on a magnet for ˜30 sec; the SDS buffer (elution) removed and saved for analysis. The elution was repeated one more time.

The samples were then analyzed by gel. 37.5 uL of sample (except elutions) was mixed with 12.5 uL 4×SDS buffer and incubated at 95° C. for 5 min. 5 uL was loaded onto a Novex 4-20% Tris-Glycine gel and run at ˜180V for ˜50 min. The gel was stained with SimplyBlue Safe Stain and imaged on a LAS4000 imager.

FIG. 94 illustrates that the affinity of NLpoly and NLpep is sufficient to allow for purification from an E. coli lysate. As NLpoly 8S was purified from an E. coli lysate, it is reasonable to expect a protein fused to NLpoly 8S (or other variant described herein) could also be purified in a similar fashion. While in this example the NLpep was immobilized and used to purify NLpoly, it is also reasonable to expect a similar result if NLpoly were immobilized.

Example 53 Kinetics of NLpoly/NLpep Binding

2× concentrations of synthetic NLpep were made and diluted 2.7-fold nine times (10 concentrations) in PBS+0.1% Prionex. Final concentrations used in the assay were 30 uM-3.9 nM. WT NLpoly (E. coli clarified lysate; 1:10,000) or 11S (1:10,000,000) was diluted in NanoGlo+100 uM Furmazine (Fz). 50 uL of NLpep was placed into wells of white 96-well assay plate. 50 uL NLpoly/NanoGlo/Fz was injected into the wells using the injector on GloMax® Multi+ instrument, and luminescence measured every 3 sec over 5 min. k_(obs) was found by fitting data to: Y=Y_(max)(1−e^(−k) ^(obs) ^(t)) using Graphpad Prism. k_(on) and k_(off) were then fitted to: k_(obs)=[NLpep]k_(on)+k_(off). FIG. 95 illustrates the association and dissociation rate constants for the binding between NLpolys and NLpeps.

Example 54 NLpoly/NLpep Substrate Affinity

NLpoly was diluted into PBS+0.1% Prionex as follows: WT at 1:10⁵, 5P at 1:10⁷, and 11S at 1:10⁸. NLpep was diluted into PBS+0.1% Prionex as follows: 30 uM for WT NLpoly studies or 3 uM for NLpoly 5P and 11S studies. 50 uL NLpoly/NLpep was incubated at RT for 5 min, 50 uL NanoGlo+Fz (ranging from 100 uM to 1.2 uM, 2×) added, and incubated for 10 min at RT. Luminescence was measured on GloMax® Multi+ with 0.5 sec integration. Km was derived using Graphpad Prism, Michaelis-Menton best-fit values. FIG. 96 illustrates the Km values for various NLpoly/NLpep pairs.

Example 55 Substrate Effect on NLpoly/NLpep Affinity

11S (E. coli clarified lysate) was diluted into PBS+0.1% Prionex at 1:10⁷. Synthetic NLpep79 was diluted serially (1:2) from 800 nM to 0.39 nM (2×). 20 uL 11S+20 uL NLpep79 were then mixed and incubated for 5 min at RT. 40 uL NanoGlo+5 uM or 50 uM Fz was added and incubated another 5 min at RT. Luminescence was measured on GloMax® Multi+ with 0.5 sec integration. Kd was derived using Graphpad prism, One site-Specific binding value. FIG. 97 illustrates that saturating concentrations of furimazine increase the affinity between 11S and NLpep79.

Example 56 Km for NLpoly 5A2:NLpep

NLpoly 5A2 was diluted into PBS+0.1% Prionex at 1:10⁵. NLpep (WT, NLpep 78 or NLpep79) was diluted into PBS+0.1% Prionex to 30 uM. 50 uL NLpoly/NLpep was incubated at RT for 5 min. 50 uL NanoGlo+Fz (ranging from 100 uM to 1.2 uM, 2×) was added and incubated for 10 min at RT. Luminescence was measured on GloMax® Multi+ with 0.5 sec integration. Km was derived using Graphpad Prism, Michaelis-Menton best-fit values. FIG. 98 illustrates the Km values for NLpoly5A2 and NLpep WT, 78, and 79.

Example 57 Luminescence of NLpoly without NLpep

E. coli clarified lysate were prepared as described previously for NLpoly WT, 5A2, 5P, 8S and 11S. 50 uL of each lysate and 50 uL NanoGlo+Fz were mixed and incubated for 5 min RT. Luminescence was measured on GloMax® Multi+ with 0.5 sec integration. FIG. 99 illustrates that the ability of the NLpoly to produce luminescence in the absence of NLpep gradually increased throughout the evolution process resulting in ˜500 fold higher luminescence for 11S than WT NLpoly.

Example 58 Improved Luminescence in E. coli Throughout Evolution Process

A single NLpoly colony of WT, 5A2, 5P, 8S or 11S was inoculated in 200 uL minimal media and grown for 20 hrs at 37° C. on shaker. 10 uL of the overnight culture was diluted into 190 uL fresh minimal media and grown for 20 hrs at 37° C. on shaker. 10 uL of this overnight culture was diluted into 190 uL auto-induction media (previously described) and grown for 18 hrs at 25° C. on shaker. The auto-induced cultures were diluted 50-fold (4 uL into 196 uL assay lysis buffer), 10 uL expression culture added to 40 uL of assay lysis buffer containing NLpep (synthetic; 1 nM; WT, NLpep78, NL79 or NLpep80) and shaken for 10 min at RT. 50 uL NanoGlo+Fz was added, and samples shaken for 5 min at RT. Luminescence was measured on a GloMax luminometer with 0.5 sec integration. FIG. 100 illustrates the improvement in luminescence from E. coli-derived NLpoly over the course of the evolution process, an overall ˜10⁵ improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

Example 59 Improved Luminescence in HeLa Cells Throughout Evolution Process

50 ng plasmid DNA expressing NLpoly WT, 5A2, 5P, 8S or 11S was transfected into HeLa cells into wells of a 12-well plate using FugeneHD. The cells were then incubated overnight at 37° C./5% CO₂. The media was replaced with 500 uL DMEM without phenol red, and the cells frozen at −80° C. for >30 min. The cells were thawed and transferred to 1.5 mL tubes. NLpep WT, NLpep78, NLpep79 or NLpep 80 (synthetic) were diluted to 10 nM in PBS+0.1% Prionex, and 25 ul mixed with 25 uL of each of the NLpoly cell lysate. The samples were shaken for 10 min at RT, and then 50 uL NanoGlo+100 uM Fz added and incubated for 5 min at RT. Luminescence was measured on a GloMax luminometer with 0.5 s integration. FIG. 101 illustrates the improvement in luminescence from HeLa-expressed NLpoly over the course of the evolution process, an overall ˜10⁵ improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

Example 60 Improved Luminescence in HEK293 Cells Throughout Evolution Process

50 ng plasmid DNA expressing NLpoly WT, 5A2, 5P, 8S or 11S was transfected into HEK293 cells into wells of a 12-well plate using FugeneHD. The cells were then incubated overnight at 37° C./5% CO₂. The media was replaced with 500 uL DMEM without phenol red, and the cells frozen at −80° C. for >30 min. The cells were thawed and transferred to 1.5 mL tubes. NLpep WT, NLpep78, NLpep79 or NLpep 80 (synthetic) were diluted to 10 nM in PBS+0.1% Prionex, and 25 ul mixed with 25 uL of each of the NLpoly cell lysate. The samples were shaken for 10 min at RT, and then 50 uL NanoGlo+100 uM Fz added and incubated for 5 min at RT. Luminescence was measured on a GloMax luminometer with 0.5 s integration. FIG. 102 illustrates the improvement in luminescence from HEK293-expressed NLpoly over the course of the evolution process, an overall ˜10⁴ improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

Example 61 Improved Binding Affinity Throughout Evolution

NLpoly WT, 5A2, 5P, 8S or 11S (E. coli clarified lysates) were diluted into PBS+0.1% Prionex as follows: WT 1:10⁴; 5A2 1:105; 5P 1:10⁶; 8S 1:10⁷; and 11S 1:10⁷. NLpepWT, NLpep78, NLpep79 or NLpep80 (synthetic) were serially into PBS+0.1% Prionex to 4× concentration. 25 uL NLpoly and 25 uL NLpep were mixed and incubated for 10 min at RT. 50 uL NanoGlo+100 uM Fz was added and incubated for 5 min at RT. Luminescence was measured on a GloMax Multi+ with 0.5 sec integration. Kd was determined using Graphpad Prism, One Site-Specific Binding, Best-fit values. FIG. 103 illustrates a 10⁴ fold improved affinity (starting affinity: NLpolyWT:NLpepWT, Kd-10 uM) of K_(d)<1 nM (NLpoly11S:NLpep86 or NLpoly11S:NLpep80) of the variants tested over wild-type.

Example 62 NLpoly Luminescence

Single NLpoly variant colonies were inoculated with 200 uL minimal media and grown for 20 hrs at 37° C. on a shaker. 10 uL of the overnight culture were diluted into 190 uL fresh minimal media and grown for 20 hrs at 37° C. on a shaker. 10 uL of this overnight culture was then diluted into 190 uL auto-induction media (previously described) and grown for 18 hrs at 25° C. on a shaker. 10 uL of this expression culture was mixed with 40 uL of assay lysis buffer (previously described) without NLpep or NLpep78-HT (1:3,860 dilution) or NLpep79-HT (1:10,000 dilution) and shaken for 10 min at RT. 50 uL of NanoGlo+Fz was added and again shake for 10 min at RT. Luminescence was measured on GloMax® luminometer with 0.5 sec integration. FIGS. 105-107 illustrate the luminescence of various NLpolys in the absence of NLpep.

Example 63 Solubility of NLpoly Variants

A single NLpoly variant colony (SEE FIG. 143) was inoculated into 5 mL LB culture and incubated at 37° C. overnight with shaking. The overnight culture was diluted 1:100 into fresh LB and incubated at 37° C. for 3 hrs with shaking. Rhamnose was added to the cultures to 0.2% and incubated 25° C. overnight with shaking. 900 ul of these overnight cultures were mixed with 100 uL 10× FastBreak Lysis Buffer (Promega Corporation) and incubated for 15 min at RT. A 75 uL aliquot (total) was removed from each culture and saved for analysis. The remaining culture from each sample were centrifuged at 14,000×rpm in a benchtop microcentrifuge at 4° C. for 15 min. A 75 uL aliquot of supernatant (soluble) was removed from each sample and saved for analysis. 25 uL of 4×SDS buffer was added to the saved aliquots and incubated at 95° C. for 5 min. 5 ul of each sample was loaded onto a 4-20% Tris-Glycine SDS gel and run at ˜190V for ˜50 min. The gel was stained with SimplyBlue Safe Stain and imaged on a LAS4000. FIG. 143 shows a protein gel of total lysates and the soluble fraction of the same lysate for the NLpoly variants.

Example 64 Dissociation Constants

NLpoly variant lysate (SEE FIG. 144; prepared as described previously) was diluted 1:10 into PBS+0.1% Prionex. 4× concentrations of NLpep78 (synthetic NLpep78) were made in PBS+0.1% Prionex. 20 uL NLpoly variant lysate and 20 uL NLpep were mixed and shaken for 10 min at RT. 40 uL NanoGlo/Fz was added and shaken for 10 min at RT. Luminescence was measured on a GloMax® luminometer with 0.5 s integration. Kd determined using Graphpad Prism, One site-specific binding, best-fit values. FIG. 144 illustrates dissociation constants of NLpep78 with various NLpolys.

Example 65 Comparison of Luminescence Generated by Cells Expressing Different Combinations of FRB and FKBP Fused to NLpoly5P and NLpep80/87

HEK293T cells (400,000) were reverse-transfected with 1 μg pF4A Ag FKBP or 1 μg pF4A Ag FRB vectors expressing N- or C-terminal fusions of NLpoly5P and/or NLpep80/87 using FuGENE HD at a DNA-to-FuGENE HD ratio of 1:4. 24-hours post transfection, cells were trypsinized and re-plated in opaque 96-well assay plates at a density of 10,000 cells per well. 24-hours after plating, cells were washed with PBS and then incubated with or without 20 nM rapamycin for 15, 60 or 120 min in phenol red-free OptiMEMI. 10 μM furimazine substrate with or without 20 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIGS. 108 (15 min induction), 109 (60 min induction) and 110 (120 min induction) illustrate a general increase in induction over time, with NLpoly5P and NLpep80 combinations generating the most luminescence. Individual components contribute minimally to signal.

Example 66 Comparison of Luminescence Generated by Cells Expressing Different Combinations of FRB and FKBP Fused to NLpoly5P and NLpep80/87

Although similar to Example 65, this example tested all 8 possible combinations of FRB and FKBP fused to NLpoly/NLpep as well as used less total DNA. HEK293T cells (400,000) were reverse-transfected with a total of 0.001 μg pF4A Ag FRB-NLpoly5P and 0.001 μg pF4A Ag FKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with 0 or 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with 0 or 50 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 111 illustrates that NLpep80 combinations generated the highest luminescence and that all configurations respond to rapamycin treatment.

Example 67 Comparison of Luminescence Generated by FRB or FKBP Fusions Expressed in the Absence of Binding Partner

HEK293T cells (400,000) were reverse-transfected with a total of 0.001 μg pF4A Ag FRB-NLpoly5P or pF4A Ag FKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with 0 or 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with 0 or 50 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 112 illustrates that the individual components generate a low basal level of luminescence that is not responsive to rapamycin treatment.

Example 68 Comparison of Luminescence Generated by Cells Transfected with Varying Amounts of FRB-NLpoly5P and FKBP-NLpep80/87 DNA

HEK293T (400,000) cells were reverse-transfected with a total of 2, 0.2, 0.02, or 0.002 μg pF4A Ag FRB-NLpoly5P and pF4A Ag FKBP-NLpep80 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 20 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 20 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 113 illustrates that transfection with less DNA decreases overall luminescence but increases fold induction.

Example 69

Comparison of Luminescence Generated by Cells Transfected with Varying Amounts of FRB-NLpoly5P or FKBP-NLpep80/87 DNA in the Absence of Binding Partner

HEK293T cells (400,000) were reverse-transfected with a total of 2, 0.2, 0.02, or 0.002 μg pF4A Ag FRB-NLpoly5P or pF4A Ag FKBP-NLpep80 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2 μg. 24-hours post-transfection, 10,000 cells were replated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 20 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 20 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 114 illustrates that lower DNA levels do not change overall luminescence of cells transfected with individual components.

Example 70 Comparison of Luminescence Generated by Cells Transfected with Varying Amounts of FRB-NLpoly5P and FKBP-NLpep80/87 DNA

HEK293T cells (400,000) were reverse-transfected with a total of 0.2, 0.02, 0.002, or 0.0002 μg pF4A Ag FRB-NLpoly5P and pF4A Ag FKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 115 illustrates that luminescence above background, as determined in Examples 69 and 71, and rapamycin induction can be achieved with DNA levels down to 2.5 pg.

Example 71 Comparison of Luminescence Generated by Cells Transfected with Varying Amounts of FRB-NLpoly5P or FKBP-NLpep80/87 DNA in the Absence of Binding Partner

HEK293T cells (400,000) were reverse-transfected with a total of 0.2, 0.02, 0.002, or 0.0002 μg pF4A Ag FRB-NLpoly5P or pF4A Ag FKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 116 illustrates no significant change in luminescence generated by individual components when less DNA was used.

Example 72 Comparison of Luminescence Generated by Cells Transfected with Varying Amounts of FRB-NLpoly5P and FKBP-NLpep80 or FKBP-NLpep87 DNA after Treatment with Rapamycin for Different Lengths of Time

HEK293T cells (400,000) were reverse-transfected with a total of 2, 0.2, 0.02, or 0.002 μg pF4A Ag FRB-NLpoly5P and pF4A Ag FKBP-NLpep80 or FKBP-NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 20 nM rapamycin for 5/15/30/60/120 min. 10 μM furimazine substrate (final concentration on cells) with or without 20 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIGS. 117 and 118 illustrates a decline in luminescence with less DNA and an increase in rapamycin induction over time.

Example 73 Comparison of Luminescence Generated by Cells Expressing Different Combinations of FRB-NLpoly5P or FRB-NLpoly5A2 with FKBP-NLpep80/87/95/96/97

In this example, the assay was performed in both a two-day and three-day format. For the 2 day assay, 20,000 HEK293T cells were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly5P or FRB-NLpoly5A2 and pF4A Ag FKBP-NLpep80/87/95/96/97 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time.

For 3 day assay, 400,000 HEK293T cells were reverse-transfected with a total of 0.002 μg pF4A Ag FRB-NLpoly5P and pF4A Ag FKBP-NLpep80/87/95/96/97 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIGS. 119 and 120 illustrate similar levels of luminescence in both the 2 day and 3 day assays. Assays performed with NLpoly5A2 showed greater rapamycin induction relative to NLpoly5P, and assays performed with NLpoly5A2 and NLpep96 showed greatest rapamycin induction of all tested combinations.

Example 73 Comparison of Luminescence Generated by Cells Expressing Different Combinations of FRB-NLpoly5A2 or FRB-NLpoly11S with FKBP-NLpep101/104/105/106/107/108/109/110

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly5A2/11S and pF4A Ag FKBP-NLpep101/104/105/106/107/108/109/110 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 121 illustrates that, of tested combinations, NLpoly11S with NLpep101 showed the greatest rapamycin induction and one of the strongest rapamycin-specific luminescent signals.

Example 74 Comparison of Luminescence Generated by Cells Transfected with Different Combinations of FRB-NLpoly5A2 or FRB-NLpoly11S with FKBP-NLpep87/96/98/99/100/101/102/103

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly5A2/11S and pF4A Ag FKBP-NLpep87/96/98/99/100/101/102/103 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 122 illustrates that the NLpoly11S and NLpep101 combination produces the highest induction while maintaining high levels of specific luminescence.

Example 75 Comparison of Luminescence Generated by Cells Transfected with Different Levels of FRB-NLpoly11S and FKBP-NLpep87/101/102/107 DNA

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.01, 0.1, 1, or 10 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep87/101/102/107 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 1.5 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 123 illustrates NLpoly11S with NLpep101 produces the overall lowest luminescence in untreated samples at all tested DNA levels, and the combination maintains relatively high levels of luminescence in rapamycin-treated samples.

Example 76 Comparison of Luminescence Generated by Cells Transfected with Different Levels of FRB-NLpoly5A2 and FKBP-NLpep87/101/102/107 DNA

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.01, 0.1, 1, or 10 ng pF4A Ag FRB-NLpoly5A2 and pF4A Ag FKBP-NLpep87/101/102/107 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 1.5 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 124 illustrates that NLpoly5A2 generates higher luminescence in untreated samples than NLpoly11S shown in example 75.

Example 77 Rapamycin Dose Response Curve Showing Luminescence of Cells Expressing FRB-NLpoly5P and FKBP-NLpep80/87 DNA

HEK293T cells (400,000) were reverse-transfected with a total of 0.001 μg pF4A Ag FRB-NLpoly5P and 0.001 μg pF4A Ag FKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with 0 to 500 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with 0 to 500 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. Kd was calculated with GraphPad Prism version 5.00 for Windows. FIG. 125 illustrates a rapamycin-specific increase in luminescence.

Example 78 Rapamycin Dose Response Curve Showing Luminescence of Cells Expressing FRB-NLpoly5A2 and FKBP-NLpep87/101 DNA

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly5A2/11S and pF4A Ag FKBP-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with 0 to 1 μM rapamycin for 1.5 h. 10 μM furimazine substrate (final concentration on cells) with 0 to 1 μM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 126 illustrates a sigmoidal dose response to rapamycin with NLpoly5A2/NLpep101 and NLpoly11S/NLpep101 combinations. While combinations with NLpep87 show an increase in luminescence with rapamycin, the collected data points deviate more from the sigmoidal curve.

Example 79 Comparison of Luminescence Generated by Cells Expressing FRB-11S and FKBP-101 and Treated with Substrate PBI-4377 or Furimazine

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1/1/10 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with 0 or 50 nM rapamycin for 1.5 h. 10 μM furimazine or PBI-4377 substrate (final concentration on cells) with 0 to 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 127 illustrates a decrease in luminescence and fold induction with the PBI-4377 substrate compared to the furimazine substrate.

Example 80 Time Course of Cells Expressing FRB-NLpoly11S/5A2 and FKBP-NLpep87/101 Conducted in the Presence or Absence of Rapamycin

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S/5A2 and pF4A Ag FKBP-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 50 nM rapamycin and 10 μM furimazine was added either manually or via instrument injection Luminescence was immediately measured on a GloMax Multi with 0.5 s integration time. FIGS. 128 and 129 illustrate that, of all combinations tested, NLpoly11S with NLpep101 has the lowest luminescence at time 0, hits a luminescent plateau faster and has the largest dynamic range.

Example 81 Luminescence Generated by FRB-NLpoly11S and FKBP-NLpep101 as Measured on Two Different Instruments

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 50 nM rapamycin was added for 20 min. 10 μM furimazine (final concentration on cells) in OptiMEMI with 0 or 50 nM rapamycin was added and incubated for an additional 5 min. Luminescence was immediately measured on a GloMax Multi with 0.5 s integration time and on the Varioskan Flash with 450 nM band pass filter. FIG. 130 illustrates that the rapamycin-specific induction of FRB-NLpoly11S and FKBP-NLpep101 can be measured on different instruments.

Example 82 Images Showing Luminescence of Cells Expressing FRB-NLpoly11S and FKBP-NLpep101 at Various Times after Treatment with Rapamycin

HeLa cells (500,000) were reverse transfected with 1 μg pF4 Ag FRB-NLpoly11S and 1 μg pF4 Ag FKBP-NLpep101 using FuGENE HD at a DNA to FuGENE ratio of 1 to 4. Cells were transfected in 35 mm glass bottom culture dishes (MatTek #p35gc-1.5-14-C). 24 hours post-transfection, cells were washed with PBS and then incubate with 10 μM furimazine in OptiMEM for 5 min. 50 nM rapamycin in OptiMEMI was added to cells and luminescent images were acquired with LV200 at 10 s intervals for a total of 20 min. Instrument was at 37° C., objective was 60×, gain was 200 and exposure was 600 ms. FIG. 131 illustrates that imaging can detect an increase in cellular luminescence in cells expressing FRB-NLpoly11S and FKBP-NLpep101 following rapamycin treatment.

Example 83 Quantitation of the Signal Generated by Individual Cells Expressing FRB-NLpoly11S and FKBP-NLpep101 at Various Times after Treatment with Rapamycin

HeLa cells (500,000) were reverse transfected with 1 μg pF4 Ag FRB-NLpoly11S and 1 μg pF4 Ag FKBP-NLpep101 using FuGENE HD at a DNA to FuGENE ratio of 1 to 4. Cells were transfected in 35 mm glass bottom culture dishes (MatTek #p35gc-1.5-14-C). 24 hours post-transfection, cells were washed with PBS and then incubate with 10 μM furimazine in OptiMEM for 5 min. 50 nM rapamycin in OptiMEMI was added to cells, and luminescent images were acquired with LV200 at 10 s intervals for a total of 20 min. Instrument was at 37° C., objective was 60×, gain was 200, and exposure was 600 ms. The signal intensity of every cell in the field of view was analyzed with Image J software over the entire time period. FIG. 132 illustrates that signal generated by individual cells can be measured and that the increase in signal by each cell parallels the increase observed in the 96-well plate assay shown in FIGS. 128 and 129.

Example 84 Comparison of Luminescence in Different Cell Lines Expressing FRB-NLpoly11S and FKBP-NLpep101

HEK293T, HeLa, or U2-OS cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 50 nM rapamycin was added for 20 min. 10 μM furimazine (final concentration on cells) in OptiMEMI with 0 or 50 nM rapamycin was added and incubated for an additional 5 min Luminescence was immediately measured on a GloMax Multi with 0.5 s integration time. FIG. 133 illustrates similar levels of luminescence generated in the absence and presence of rapamycin in three different cells lines transfected with FRB-NLpoly11S and FKBP-NLpep101.

Example 85 Comparison of Luminescence Generated by Cells Expressing FRB-NLpoly11S and FKBP-NLpep101 after Treatment with the Rapamycin Competitive Inhibitor FK506

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 20 nM rapamycin was added for 20 min. FK506 inhibitor in OptiMEM was added to cell at final concentration of 5 μM and incubated for 3 or 5 hours. Furimazine in OptiMEM was added to cells for a final concentration of 10 μM on cells Luminescence was immediately measured on a GloMax Multi with 0.5 s integration time. FIG. 134 illustrates a decrease in rapamycin-induced luminescence after treatment with the competitive inhibitor FK506.

Example 86 Luminescence Generated by Cells Expressing FRB-NLpoly11S and FKBP-NLpep101 after Treatment with the Rapamycin Competitive Inhibitor FK506

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 20 nM rapamycin was added for 2.5 hours. FK506 inhibitor in OptiMEM was added to cell via injector at final concentration of 0, 1 or 10 μM in OptiMEM with 10 μM Luminescence was measured every 10 min for 4 hours on a GloMax Multi set to 37° C. with 0.5 s integration time. FIG. 135 illustrates that by 200 s, FK506 inhibitor can reduce luminescence close to levels of untreated cells.

Example 87 Luminescence Generated by Cells Transfected with Different Combinations of V2R-NLpoly5A2 or V2R-NLpoly11S with NLpep87/101-ARRB2 in the Presence or Absence of the V2R Agonist AVP

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1, 1, or 10 ng pF4A Ag V2R-NLpoly11S and pF4A Ag ARRB2-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 1 μM AVP and 10 μM furimazine was added for 25 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 136 illustrates that V2R-NLpoly11S with NLpep101 generates the greatest AVP-specific increase in luminescence. Combinations with NLpep87 show no significant response to AVP.

Example 88 Time Course Showing Luminescence Generated by Cells Transfected with V2R-NLpoly5A2 or V2R-NLpoly11S and NLpep87/101-ARRB2 after Treatment with AVP

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 or 1 ng pF4A Ag V2R-NLpoly11S or 1 ng pF4A Ag V2R-NLpoly5A2 and pF4A Ag ARRB2-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 1 μM AVP and 10 μM furimazine was added either manually (FIG. 137) or via instrument injection (FIG. 138). Luminescence was then measured on a GloMax Multi every 5 min for 25 min with 0.5 s integration time at room temperature (FIGS. 137 and 138) or 37° C. (FIG. 139). FIGS. 137 and 138 illustrate a time-dependent increase in AVP-induced luminescence for V2R-NLpoly11S with NLpep101-ARRB2 that begins to peak at 600 s. Combinations with V2R-NLpoly5A2 and NLpep87 do not show a significant increase in luminescence over time. FIG. 139 illustrates that at 37° C. all NLpoly11S and NLpep101 combinations tested show a time-dependent increase in AVP-induced luminescence that levels out around 200 s.

Example 89 Comparison of Luminescence in Different Cell Lines Expressing V2R-NLpoly11S and NLpep101-ARRB2

HEK293T, HeLa, or U2-OS cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 1 ng pF4A Ag V2R-NLpoly11S and pF4A Ag ARRB2-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 1 μM AVP was added for 20 min. Furimazine in OptiMEM was then added to a final concentration of 10 μM on cells, and luminescence was measured on a GloMax Multi with 0.5 s integration time.

FIG. 140 illustrates similar luminescence levels in three different cell lines expressing V2R-NLpoly11S and NLpep101-ARRB2 in the presence and absence of AVP.

Example 90 Luminescence of Cells Expressing V2R-NLpoly11S and NLpep101-ARRB2 at Various Times after Treatment with AVP

HeLa cells (500,000) were reverse transfected with 1 μg pF4 Ag V2R-NLpoly11S and 1 μg pF4 Ag ARRB2-NLpep101 using FuGENE HD at a DNA to FuGENE ratio of 1 to 4. Cells were transfected in 35 mm glass bottom culture dishes (MatTek #p35gc-15-14-C). 24 hours post-transfection, cells were washed with PBS and then incubate with 10 μM furimazine in OptiMEM for 5 min. 1 μM AVP in OptiMEMI was added to cells, and luminescent images were acquired with LV200 at 15 s intervals for a total of 30 min. Instrument was at 37° C., objective was 60× or 150×, gain was 600, and exposure was is or 2 s. FIGS. 141 and 142 illustrate that imaging can detect the increase in luminescence and formation of punctuate in individual cells after treatment with AVP.

Example 91 Dissociation Constants for NLpeps

NLpoly 5P E. coli clarified lysate (prepared as described previously) was diluted 1:1,000 into PBS+0.1% Prionex. 4× concentrations of NLpep78-HT (E. coli clarified lysate prepared as described previously) were made in PBS+0.1% Prionex. 20 uL NLpoly 5P and 20 uL NLpep78 were mixed and shaken for 10 min at RT. 40 uL NanoGlo/Fz was added and shaken for 10 min at RT. Luminescence was measured on GloMax luminometer with 0.5 s integration. Kd was determined using Graphpad Prism, One site-specific binding, best-fit values. FIG. 80 compares the dissociation constants for an NLpep consisting of either 1 or 2 repeat units of NLpep78.

Example 92 Affinity Between NLpoly 5A2 and NLpep86

NLpoly 5A2 lysate (prepared as described previously after transfecting CHO cells) was diluted 1:10 into PBS+0.1% Prionex. 4× concentrations of NLpep86 (synthetic NLpep) were made in PBS+0.1% Prionex. 20 uL NLpoly and 20 uL NLpep were mixed and shaken for 10 min at RT. 40 uL NanoGlo/Fz was added and shaken for 10 min at RT. Luminescence was measured on GloMax luminometer with 0.5 s integration. Kd was determined using Graphpad Prism, One site-specific binding, best-fit values. FIG. 81 illustrates the affinity between NLpoly 5A2 and NLpep86.

Example 93 Luminescence of NLpoly Variants

A single colony of various NLpolys were inoculated individually into 200 uL minimal media and grown for 20 hrs at 37° C. on shaker. 10 uL of overnight culture was diluted into 190 uL fresh minimal media and grown for 20 hrs at 37° C. on shaker. 10 uL of this overnight culture was diluted into 190 uL auto-induction media (previously described) and grow for 18 hrs at 25° C. on shaker. 10 uL of the expression culture was mixed with 40 uL of assay lysis buffer (previously described) without NLpep or with NLpep78-HT (1:3,860 dilution) or NLpep79-HT (1:10,000 dilution). The mixtures were shaken for 10 min at RT, 50 uL NanoGlo+Fz added and shaken again for 10 min at RT. Luminescence was measured on a GloMax luminometer with 0.5 sec integration. FIG. 82 demonstrates the luminescence from NLpoly variants without an NLpepa NLpep or with NLpep78 or NLpep79. The results show that the NLpoly variant 11S (12S-51) has improved luminescence over the other variants.

Example 94 Dissociation Constants and Vmax Values for NLpolys with 96 Variants of NLpeps

NLpeps were synthesized in array format by New England Peptide (peptides blocked at N-terminus by acetylation and at C-terminus by amidation; peptides in arrays were synthesized at ˜1 mg scale). Each peptide was lyophilized in 3 separate plates. Each well from 1 of the 3 plates of peptides was dissolved in 100 uL nanopure water, and the A260 measured and used to calculate the concentration using the extinction coefficient of each peptide. The concentration was then adjusted based on the purity of the peptide, and nanopure water was added to give a final concentration of 750 uM.

Peptides were diluted to 12.66 uM (4×) in PBS+0.1% Prionex and then diluted serially 7 times (8 concentrations total) in 0.5 log steps (3.162 fold dilution). NLpolys 5P, 8S, 5A2 or 11S were diluted into PBS+0.1% Prionex as follows: 5P 1:2,000; 8S 1:10,000; 11S 1:150,000, 5A2 1:1,000. 25 uL each NLpep+25 uL each NLpoly were mixed and incubated for 30 min at RT. 50 uL NanoGlo+100 uM Fz was added and incubated for 30 min at RT. Luminescence was measure on a GloMax Multi+ with 0.5 sec integration. Kd/Vmax were determined using Graphpad Prism, One site-specific binding, best-fit values. FIGS. 83-90 illustrate the dissociation constant and Vmax values from NLpolys with the 96 variant NLpeps. The results indicate specific mutations in the NLpeps that exhibit lower binding affinity without loss in Vmax.

Example 95 Solubility of NLpoly Variants

A single NLpoly 5A2, 12S, 11S, 12S-75, 12S-107 or 5P-B9 colony was inoculated into 5 mL LB culture and incubated at 37° C. overnight with shaking. The overnight culture was diluted 1:100 into fresh LB and incubated at 37° C. for 3 hrs with shaking. Rhamnose was added to the cultures to 0.2% and incubated 25° C. overnight with shaking. 900 ul of these overnight cultures were mixed with 100 uL 10× FastBreak Lysis Buffer (Promega Corporation) and incubated for 15 min at RT. A 75 uL aliquot (total) was removed from each culture and saved for analysis. The remaining culture from each sample was centrifuged at 14,000×rpm in a benchtop microcentrifuge at 4° C. for 15 min. A 75 uL aliquot of supernatant (soluble) was removed from each sample and saved for analysis. 25 uL of 4×SDS buffer was added to the saved aliquots and incubated at 95° C. for 5 min. 5 ul of each sample was loaded onto a 4-20% Tris-Glycine SDS gel and run at ˜190V for ˜50 min. The gel was stained with SimplyBlue Safe Stain and imaged on a LAS4000. FIG. 91 shows a protein gel of total lysates and the soluble fraction of the same lysate for the NLpoly variants. With the exception of 5A2, all variants exhibit a percentage of NLpoly in the soluble fraction.

Example 96 Solubility and Dissociation Constant of NLpoly Variants

A single NLpoly colony (listed in FIG. 92) was inoculated into 5 mL LB culture and incubated at 37° C. overnight with shaking. The overnight culture was diluted 1:100 into fresh LB and incubated at 37° C. for 3 hrs with shaking. Rhamnose was added to the cultures to 0.2% and incubated 25° C. overnight with shaking. 900 ul of these overnight cultures were mixed with 100 uL 10× FastBreak Lysis Buffer (Promega Corporation) and incubated for 15 min at RT. A 75 uL aliquot (total) was removed from each culture and saved for analysis. The remaining culture from each sample was centrifuged at 14,000×rpm in a benchtop microcentrifuge at 4° C. for 15 min. A 75 uL aliquot of supernatant (soluble) was removed from each sample and saved for analysis. 25 uL of 4×SDS buffer was added to the saved aliquots and incubated at 95° C. for 5 min. 5 ul of each sample was loaded onto a 4-20% Tris-Glycine SDS gel and run at ˜190V for ˜50 min. The gel was stained with SimplyBlue Safe Stain and imaged on a LAS4000. FIG. 92 shows a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants as well a table containing the dissociation constants for the same variants.

Example 97 Substrate Specificity for NLpoly 5P and 11S with NLpep79

E. coli clarified lysates were prepared for NLpoly 5P or 11S as described previously. The NLpoly lysates were then serially diluted in steps of 10-fold into PBS+0.1% Prionex. 25 uL NLpoly and 25 uL synthetic NLpep79 (400 nM, 4×) were mixed and incubated for 10 min at RT. 50 uL NanoGlo+100 uM Fz was added, incubated for 10 min at RT, luminescence measured on a GloMax Multi+ with 0.5 sec integration. FIG. 93 shows the substrate specificity for 5P and 11S with NLpep79 and demonstrates that 11S has superior specificity for furimazine than 5P.

Example 98 Solubility of NLpoly variants from Various Steps of Evolution

A single NLpoly WT, 5A2, 5P, 8S or 11S colony was inoculated into 5 mL LB culture and incubated at 37° C. overnight with shaking. The overnight culture was diluted 1:100 into fresh LB and incubated at 37° C. for 3 hrs with shaking. Rhamnose was added to the cultures to 0.2% and incubated 25° C. overnight with shaking. 900 ul of these overnight cultures were mixed with 100 uL 10× FastBreak Lysis Buffer (Promega Corporation) and incubated for 15 min at RT. A 75 uL aliquot (total) was removed from each culture and saved for analysis. The remaining culture from each sample was centrifuged at 14,000×rpm in a benchtop microcentrifuge at 4° C. for 15 min. A 75 uL aliquot of supernatant (soluble) was removed from each sample and saved for analysis. 25 uL of 4×SDS buffer was added to the saved aliquots and incubated at 95° C. for 5 min. 5 ul of each sample was loaded onto a 4-20% Tris-Glycine SDS gel and run at ˜190V for ˜50 min. The gel was stained with SimplyBlue Safe Stain and imaged on a LAS4000. FIG. 104 shows a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants from various steps of the evolution process. These results demonstrate that the solubility of NLpoly was dramatically increased in the evolution process.

Example 99 Chemical Labeling of Proteins

The non-luminescent peptides (NLpeps) of the present invention can be used to chemically label proteins. A NLpep of the present invention can be synthesized to contain a reactive group, e.g., biotin, succinimidyl ester, maleimide, etc., and attached (e.g., conjugated, linked, labeled, etc.) to a protein, e.g., antibody. The NLpep-labeled protein, e.g., NLpep-antibody, can then be used in a variety of applications, e.g., ELISA. The interaction/binding of the NLpep-labeled protein, e.g., NLpep-antibody, to its target/binding partner would be detected by adding a NLpoly of the present invention and NanoGlo® assay reagent. The luminescence generated by the interaction of the NLpep-labeled protein and NLpoly would correlate to the interaction of the NL-labeled protein to its target/binding partner. This concept could allow for multiple NLpeps to be attached to a single protein molecule thereby resulting in multiple NLpep-labeled protein/NLpoly interactions leading to signal amplification.

Example 100 Detection of Post-Translational Protein Modification Using HaloTag-NLpep by Western Blotting

Several proteins can be posttranslationally modified by AMPylation or ADP-ribosylation. In AMPylation, AMP is added to the target protein by a phosphodiester bond using ATP as the donor molecule. Similarly, in ADP-ribosylation, an ADP-ribose moiety is added to target proteins through a phosphodiester bond using NAD+ as the donor molecule. It has been shown that the N6-position of both ATP and NAD+ can be used to tag linkers without affecting the posttranslational event. If a N6-modified chloroalkane-ATP or -NAD+ is used to perform the AMPylation or ADP-ribosylation reaction, the target proteins would be modified to contain the chloroalkane-ATP or -NAD+.

The N6-modified ATP/NAD has been used in combination with click-chemistry to develop in-gel fluorescent-based detection systems. Detection of these post-translational modifications by western blotting techniques requires antibodies, which are often not specific or not available. An alternative approach could be to combine the properties of HaloTag® technology and the high luminescence of NanoLuc® luciferase (NL).

Upon post-translational modification of target proteins with chloroalkane-ATP (for AMPylation) or chloroalkane-NAD+ (for ADP-ribosylation) using either cell lysate or purified proteins, samples can be resolved by SDS-PAGE and transferred to PVDF membrane. Following blocking, the blot can be incubated with HaloTag-NLpep. HaloTag will bind to the post-translationally-modified proteins. In the next step, the NLpoly and furimazine could be added to the blot to detect the bioluminescence. This detection method is an alternative to a chemiluminescent-based approach for detection of western blots. A chemiluminescent-based approach could involve incubation HaloTag-protein G fusions (as a primary) in the next step any secondary antibody-linked to HRP could be used followed by ECL reaction.

Example 101 Post Translational Modification Assays

Post translational modifications (PTMs) of proteins are central to all aspects of biological regulation. PTMs amplify the diverse functions of the proteome by covalently adding functional groups to proteins. These modifications include phosphorylation, methylation, acetylation, glycosylation, ubiquitination, nitrosylation, lipidation and influence many aspects of normal cell biology and pathogenesis. More specifically, histone related PTMs are of great importance. Epigenetic covalent modifications of histone proteins have a strong effect on gene transcriptional regulation and cellular activity. Examples of post translational modification enzymes include but not limited to, Kinases/Phosphatases, Methyltransferases (HMT)/Demethylases (HDMT), Acetyltransferases/Histone Deacetylases, Glycosyltransferases/Glucanases and ADP-Ribosyl Transferases. Under normal physiological conditions, the regulation of PTM enzymes is tightly regulated. However, under pathological conditions, these enzymes activity can be dysregulated, and the disruption of the intracellular networks governed by these enzymes leads to many diseases including cancer and inflammation.

The non-luminescent peptides (NLpep) and non-luminescent polypeptides (NLpoly) of the present invention can be used to determine the activity of PMT enzymes by monitoring changes in covalent group transfer (e.g. phosphoryl, acetyl) to a specific peptide substrate linked to a NLpep of the present invention. The NLpep will be linked through peptide synthesis to small PTM enzyme specific peptide and used as a substrate for the PTM enzyme.

A) PTM Transferase Assays (HAT)

Once the PTM enzyme reaction has occurred, an aminopeptidase can be used to degrade the non-modified peptide (NLpep; control). The methylatedmodified (acetylated) peptide (NLpep-PTM enzyme substrate) would be degraded at a very slow rate or would not be degraded at all as the aminopeptidase activity is known not to degrade to be affected by a PTM. Once the aminopeptidase reaction is complete, the NLpoly is added with the NanoGlo® assay reagent containing Furimazine Luminescence would be generated from the sample where PTM occurred via the interaction of the NLpep and NLpoly. If no PTM occurred, the NLpep would be degraded, and no interaction between the NLpep and NLpoly would occur, thereby no luminescence would be generated. This concept is exemplified in FIG. 145 for H3K4/9 acetyltransferases.

The reaction would be performed under optimal enzyme reaction condition using the histone peptide substrate linked to NLpep of the present invention and Acetyl-CoA or SAM as the acetyl or methyl group donor. A buffer containing aminopeptidase or a mixture of aminopeptidases would be added to degrade specifically all the non-modified substrates. A buffer containing a NLpoly of the present invention and an aminopeptidase inhibitor would be added. NanoGlo® assay reagent would be added, and luminescence detected. Luminescence generated would be proportional to the amount of non-degraded NLpep present, and therefore would correlate with the amount of methylated or acetylated substrates, thereby indicating the amount of methyl or acetyl transferase activity. The assay can also be applied to PTM such as phosphorylation, glycosylation, ubiquitination, nitrosylation, and lipidation.

B) PTM Hydrolase Assays (HDMT)

In a similar concept to A) can be used for Histone Demethylases (HDMT). However, instead of an aminopeptidase, a PMT-specific antibody can be used to create activity interference. An NLpepA NLpep of the present invention could be linked through peptide synthesis to small methylated peptide and used as a substrate for the hydrolase. Once a hydrolase reaction has been completed, an anti-methyl antibody can be added to the reaction. This antibody will bind specifically to the methylated peptide (control). The peptide product generated by the HDMT will not bind to the antibody. Then, an NLpoly of the present invention can be added. If the antibody interferes with the interaction of NLpep and NLpoly, no luminescence will be generated. If there was hydrolysis of the PTM by the demethylase, the NLpep and NLpoly will interact, and luminescence will be generated. This concept is exemplified in FIG. 146 H3K4/9 demethylases.

The concept of aminopeptidase degradation of the non-modified substrate can also be used for a hydrolase assay except it would be a loss of signal assay instead of a gain of signal. The reaction would be performed under optimal enzyme reaction condition using a modified (methylated or acetylated) histone peptide substrate linked to an NLpepa NLpep of the present invention. A buffer containing an antibody capable of recognizing the methyl or acetyl group would be added. A buffer containing an NLpolya NLpoly of the present invention would be added. The NLpoly would interact with NLpep not bound to the antibody. NanoGlo® assay reagent would be added, and luminescence detected. The luminescence generated would be proportional to the amount of NLpep not bound to the antibody, and therefore would correlate with the amount of demethylated or deacetylated substrate, thereby indicating the amount of demethylase or deacetylase activity. Both hydrolase assay concepts can also be applied to PTM hydrolases such as phosphatases, glucanases and deubiquitinases.

In another version of these concepts, the PTM transfer or hydrolysis on the peptide-NLpep would be alone sufficient to reduce or enhance the interaction of NLpep with NLpoly and therefore decrease or increase the luminescence signal without the need of aminopeptidase or antibody.

Example 102 Detection of Specific RNAs (Noncoding RNA or mRNA) of Interest in Mammalian Cells, Cell Lysate or Clinical Sample

The non-luminescent peptide (NLpep) and non-luminescent polypeptide (NLpoly) of the present invention can be tethered to an RNA binding domain (RBD) with engineered sequence specificity. The specificity of the RBD can be changed with precision by changing unique amino acids that confers the base-specificity of the RBD. An example of one such RBD is the human pumilio domain (referred here as PUM). The RNA recognition code of PUM has been very well established. PUM is composed of eight tandem repeats (each repeat consists of 34 amino acids which folds into tightly packed domains composed of alpha helices). Conserved amino acids from the center of each repeat make specific contacts with individual bases within the RNA recognition sequence (composed of eight bases). The sequence specificity of the PUM can be altered precisely by changing the conserved amino acid (by site-directed mutagenesis) involved in base recognition within the RNA recognition sequence. For detection of specific RNAs in the cell, PUM domains (PUM1 and PUM2) with customized sequence specificities for the target RNA can be tethered to a NLpep and NLpoly of the present invention (e.g., as a genetic fusion protein via genetic engineering) and can be expressed in mammalian cells. PUM1 and PUM2 are designed to recognize 8-nucleotide sequences in the target RNA which are proximal to each other (separated by only few base pairs, determined experimentally). Optimal interaction of PUM1 and PUM2 to their target sequence is warranted by introducing a flexible linker (sequence and length of the linker to be determined experimentally) that separates the PUM and the non-luminescent peptide and non-luminescent polypeptide. Binding of the PUM1 and PUM2 to their target sequence will bring the NLpep and NLpoly into close proximity in an orientation that results in a functional complex formation capable of generating bioluminescent signal under our specific assay condition. A functional bioluminescent complex would not be generated in the absence of the RNA target due to the unstable interaction of the NLpep and NLpoly pairs that constitutes the complex.

A similar strategy can also be used for detecting RNA in clinical sample in vitro. The NLpep-PUM fusion proteins with customized RNA specificity can be expressed and purified from suitable protein expression system (such as E. coli or mammalian protein expression system). Purified components can be added to the biological sample along with suitable substrate and assay components to generate the bioluminescent signal.

Example 103 DNA Oligo-Based Detection of Specific RNA (Noncoding RNA or mRNA) in Clinical Sample or Mammalian Cell Lysate

A non-luminescent peptide (NLpep) and non-luminescent polypeptide (NLpoly) of the present invention can be attached to oligonucleotides complementary to the target RNA with suitable linker (amino acids or nucleotides). Functional assembly of bioluminescent complex occurs only when sequence specific hybridization of DNA oligo to their target RNA brings the NLpep and NLpoly into close proximity in an ideal conformation optimal for the generation of a bioluminescent signal under the assay conditions. The detection can also be achieved through a three-component complementation system involving two NLpeps and a third NLpoly. For example, two NLpep-DNA conjugates will be mixed with the target RNA. Functional assembly of the bioluminescent complex is achieved by subsequent addition of the third NLpoly. Thus, if a detectable signal is produced under specific assay conditions using a clinical sample or cell lysate, the presence of target RNA in such a sample is inferred. Such assays are useful for detecting RNAs derived from infectious agents (viral RNAs) and specific RNA biomarkers (implicated in many disease conditions such as various forms of cancers, liver diseases, and heart diseases), and could provide a new avenue for diagnosis and prognosis of many disease conditions.

Example 104 In-Vivo Imaging

Biotechnology-derived products (Biologics), including antibodies, peptides and proteins, hold great promises as therapeutics agents. Unlike small molecule drugs, biologics are large molecules with secondary and tertiary structures and often contain posttranslational modifications. Internalization, intracellular trafficking, bio-distribution, pharmacokinetics and pharmacodynamics (PK/PD), immunogenicity, etc. of biologics differ significantly from small molecule drugs, and there is a need for new tools to ‘track’ these antibodies in vivo. Conventional chemical labeling with enzyme reporters (HRP, luciferase, etc.) or small fluorescent tags can significantly alter the therapeutic value of the biologics and are not ideal for in vivo imaging using biologics. Radioisotope-labeling for PET-based imaging is also not convenient.

The NLpolys and NLpeps described herein offer a novel solution for in vivo imaging of biologics. The NLpep can be genetically encoded into a biologic therapeutics without any synthetic steps. Genetic encoding allows precise control over amount of peptide per biologic molecule as well as its position, thereby minimizing any perturbation to its therapeutic value. For imaging, a NLpoly along with substrate, e.g., furimazine, can be injected into the animal. If the NLpep-biologic and NLpoly interact, luminescence would be generated. Alternatively, a transgenic animal expressing NLpoly can be used as a model system.

Example 105 BRET Applications

This concept fundamentally measures three moieties coming together. Two of the NLpolys and/or NLpeps form a complex, and the third moiety, which is either fluorescent or bioluminescent, provides an energy transfer component. If the complex formed is bioluminescent, both bioluminescence and energy transfer (i.e., BRET) can be measured. If the complex formed is fluorescent, the magnitude of energy transfer can be measured if the third component is a bioluminescent molecule.

A) This example demonstrates a fluorescent dye attached to a NLpep. Alternatively, a fluorescent protein could be fused, e.g., a fusion protein, with a NLpoly or NLpep (created from a genetic construct).

E. coli clarified lysate of NLpoly WT was prepared as described previously. 40 uL NLpoly WT lysate was mixed with 10 uL of PBI-4730 (NLpep1) or PBI-4877 (NLpep1-TMR) and incubated for 10 min at RT. 50 uL 100 uM furimazine in 50 mM HEPES pH 7.4 was added and incubated for 30 min at RT. Luminescence was measured over 400-700 nm on TECAN M1000.

FIG. 147 illustrates very efficient energy transfer from the NLPoly/NLPep complex (donor) to TMR (acceptor), and the corresponding red shift in the wavelength of light being emitted.

B) This example demonstrates using the BRET in detection, such as detecting small molecule concentration or enzymatic activity. Because energy transfer is strongly dependent on distance, the magnitude of energy transfer can often be related to the conformation of the system. For instance, insertion of a polypeptide that chelates calcium can be used to measure calcium concentration through modulation of energy transfer.

An enzyme that also changes the distance, either through causing a conformational change of the sensor as above or through cleavage of the sensor from the fluorescent moiety, can be measured through a system as described herein. A NLpoly or NLpep bound to a fluorescent moiety gives energy transfer when the NLpoly and NLpep interact. One example of this is a peptide sensor that has been made wherein the NLpep is conjugated to a fluorescent TOM dye via a DEVD linker (Caspase-3 cleavage site). When exposed to the NLpoly, energy transfer is observed. When exposed to Caspase-3, energy transfer is eliminated, but luminescence at 460 nm remains.

NLpoly 5A2 and NL-HT (NanoLuc fused to HaloTag) were purified. 20 uL of 8 μM NL-HT was mixed with 20 uL of 100 nM PBI-3781 (See, e.g., U.S. patent application Ser. No. 13/682,589, herein incorporated by reference in its entirety) and incubated for 10 min at RT. 40 uL NanoGlo+100 uM furimazine was added, and luminescence measured over 300-800 nm on TECAN M1000.

20 uL of 33 ng/uL NLpoly 5A2 was mixed with 20 uL of ˜500 uM PBI-5074 (TOM-NCT-NLpep). 40 uL NanoGlo+100 uM furimazine was added, and luminescence measured over 300-800 nm on TECAN M1000.

FIG. 148 illustrates energy transfer from the NLPoly/NLPep complex (donor) to TOM-dye (acceptor), and the corresponding red shift in the wavelength of light being emitted.

C) Ternary Interactions

The energy transfer with an NLpoly and NLpep can also be used to measure three molecules interacting. One example would be a GPCR labeled with NLpoly and a GPCR interacting protein with NLpep that forms a bioluminescent complex when they interact. This allows measurement of the binary interaction. If a small molecule GPCR ligand bearing an appropriate fluorescent moiety for energy transfer interacts with this system, energy transfer will occur. Therefore, the binary protein-protein interaction and the ternary drug-protein-protein interaction can be measured in the same experiment. Also, the fluorescent molecule only causes a signal when interacting with a protein pair, which removes any signal from the ligand interacting with an inactive protein (FIG. 149).

Example 106 6-Tetramethylrhodamine-PEG3-NH₂

To a solution of 6-Tetramethylrhodamine succidimidyl ester (0.25 g, 0.5 mmol) in DMF (5 mL), 1-Boc-4,7,10-trioxamidecan-1,13-diamine (0.15 g, 0.5 mmol) was added followed by diisopropylethylamine (0.25 mL, 1.4 mmol). After stirring for 16 h, the reaction was analyzed by HPLC to confirm complete consumption of the 6-tetramethylrhodamine succidimyl ester. The reaction was concentrated to a pink film, which was dissolved in a combination of triisopropylsilane (0.2 mL) and trifluoroacetic acid (4 mL). The pink solution was stirred for 2 h, after which analytical HPLC confirmed complete consumption of starting material. The reaction was concentrated to dryness to provide crude 6-Tetramethylrhodamine-PEG3-NH2 as a pink film.

The fully protected peptide Boc-GVTGWRLCERILA-resin was synthesized by standard solid phase peptide synthesis using Fmoc techniques, then cleaved from the resin using dichloroacetic acid to liberate the fully protected peptide as a white solid. To a solution of 6-Tetramethylrhodamine-PEG3-NH2 (0.05 g, 0.08 mmol) in DMF (1.5 mL), this Boc-GVTGWRLCERILA-OH (0.2 g, 0.07 mmol), 1-hydroxyazabenzotriazole (11 mg, 0.08 mmol), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (15 mg, 0.08 mmol) and diisopropylethylamine (0.28 mL, 0.16 mmol) was added. After stirring for 30 min, the reaction was concentrated, and the resulting crude was partitioned between CH₂Cl₂ and water, the layers separated and the organic layer was washed with water and brine, dried over sodium sulfate and concentrated. The resulting pink solid was dissolved in a combination of triisopropylsilane (0.2 mL) and trifluoroacetic acid (4 mL). After stirring for 3 h, the reaction was concentrated, and the resulting pink film was purified with reverse phase HPLC using a gradient of ACN in 0.1% aqueous TFA to provide PBI 4877 as a pink powder: MS (M+) calcd 2088.5. found 2089.1.

The fully protected peptide H-DEVDGVTGWRLCERILA-resin was synthesized by standard solid phase peptide synthesis using Fmoc techniques. While still on the resin, a solution of 6-TOM (PBI-3739) succidimidyl ester was added and allowed to react with the free N-terminus. The peptide was then cleaved from the resin and fully deprotected using trifluoroacetic acid (TFA) to provide a blue solid. This solid was purified with reverse phase HPLC using a gradient of ACN in 0.1% aqueous TFA to provide PBI 5074 as a blue powder: MS (M+Z/2) calcd 1238.9. found 1238.8.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the present invention. 

1. A peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced in the presence of a substrate when the peptide contacts a polypeptide consisting of SEQ ID NO:
 440. 2. The peptide of claim 1, wherein the detectable bioluminescent signal is produced in the presence of a substrate when the peptide associates with the polypeptide consisting of SEQ ID NO:
 440. 3. The peptide of claim 1, wherein the peptide exhibits enhancement of one or more traits compared to a peptide of SEQ ID NO: 2, wherein the traits are selected from: affinity for the polypeptide consisting of SEQ ID NO: 440, expression, intracellular solubility, intracellular stability, and bioluminescent activity when combined with the polypeptide consisting of SEQ ID NO:
 440. 4. The peptide of claim 1, wherein the amino acid sequence is selected from the peptides of Table
 1. 5. The peptide of claim 1, wherein the amino acid sequence is synthetic, contains non-natural amino acids or is a peptide mimic.
 6. A nucleic acid comprising a sequence coding for a peptide of claim
 1. 7. A fusion polypeptide comprising the peptide of claim 1 and a first interaction polypeptide that is configured to form a complex with a second interaction polypeptide upon contact of the first interaction polypeptide and the second interaction polypeptide.
 8. A nucleic acid comprising a sequence coding for a fusion polypeptide of claim
 7. 9. A bioluminescent complex comprising: (a) the fusion polypeptide of claim 7; and (b) a second fusion polypeptide comprising: (i) the second interaction polypeptide, and (ii) a complement polypeptide that emits a detectable bioluminescent signal in the presence of a substrate when associated with the peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2; wherein the first fusion polypeptide and second fusion polypeptide are associated; and wherein the peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2 and the complement polypeptide are associated. 10.-17. (canceled)
 18. A bioluminescent complex comprising: (a) a peptide comprising a peptide amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2; and (b) a polypeptide comprising a polypeptide amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 440, wherein the bioluminescent complex exhibits detectable luminescence.
 19. The bioluminescent complex of claim 18, wherein the peptide amino acid sequence and the polypeptide amino acid sequence are associated.
 20. The bioluminescent complex of claim 18, wherein the peptide amino acid sequence is selected from the peptide sequences of Table
 1. 21. The bioluminescent complex of claim 18, wherein the polypeptide amino acid sequence is selected from the polypeptide sequences of Table
 2. 22. The bioluminescent complex of claim 19, wherein the peptide amino acid sequence is attached to a first interaction element that is associated with a second interaction element.
 23. The bioluminescent complex of claim 22, wherein the polypeptide amino acid sequence is attached to the second interaction element.
 24. The bioluminescent complex of claim 23, wherein the polypeptide amino acid sequence and peptide amino acid sequence are incapable of associating in the absence of the association of the first and second interaction elements. 25.-40. (canceled)
 41. A detection reagent comprising: (a) a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced, in the presence of a substrate, when the peptide contacts a polypeptide consisting of SEQ ID NO: 440, and (b) a substrate for a bioluminescent complex produced by said peptide and a polypeptide consisting of SEQ ID NO:
 440. 